SCHOOL OF BIO AND CHEMICAL ENGINEERING

DEPARTMENT OF BIOTECHNOLOGY

UNIT – I – GENETIC ENGINEERING – SBB2102

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 rDNA TECHNOLOGY AND TOOLS INVOLVED IN GENETIC MANIPULATIONS

1. Recombinant DNA Technology:

Through the years, scientists have studied the life and how it changes and adapts to the
changes in the surroundings. One of their studies to cope up with the changes is through genetic
modification. This process has been done indirectly to plants and animals to control their
characteristics. Genetic modification is the process of changing the genetic makeup of an organism
to express new traits. Modern biotechnology has made it much easier and faster in targeting
specific genes to alter through genetic engineering. One of the most known technologies used in
genetic engineering is Recombinant DNA Technology.
DNA exists in the cells of all living things. These long chains of amino acids serve as the
genetic blueprints for living organisms. DNA controls how they form before birth and which traits
they pass on to the next generation. Recombinant DNA exists in a laboratory by combining genetic
material from multiple sources. Recombinant DNA technology can create new kinds of living
organisms or alter the genetic code of existing organisms. As with most technology, there are great
benefits and notable downsides to the use of recombinant DNA technology.
1.1.Pros of Recombinant DNA Technology
Recombinant DNA technology, sometimes referred to as "genetic engineering," can benefit
people in several ways. For example, scientists made artificial human insulin with the help of
recombinant DNA technology. Diabetic people cannot produce their own insulin, which they need
in order to process sugar. Animal insulin is not a suitable replacement, since it causes severe
allergic reactions in most people. Thus, scientists used recombinant DNA technology to isolate the
gene for human insulin and insert it into plasmids (cellular structures that can replicate
independently of chromosomes). These plasmids were then inserted into bacterial cells, which
created insulin based on the human genetic code inside of them. The resulting insulin was safe for
humans to use. Thus, people with diabetes went from having a life expectancy of around 4 years
after diagnosis to having a normal human life expectancy.
Recombinant DNA technology helped improve food production. Fruits and vegetables,
which were prone to attacks from pests, now have genetic modifications to be more resistant. Some
foods have modifications for longer shelf lives or higher nutritional content. These advancements
greatly increased crop yields, which means that more food is available to the public at the end of
each growing cycle.
Scientists have been working to improve vaccines and produce new ones using
recombinant DNA technology. These "DNA vaccines," which utilize recombinant DNA, are in the
testing stages. Most modern vaccines introduce a small "piece" of a disease into the body, so the
body can develop ways to fight that particular disease. DNA vaccines would directly introduce the
antigen itself and lead to more immediate and permanent immunity. Such vaccines could
potentially protect people against diseases such as diabetes and even cancer.

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1.2.Cons of Recombinant DNA Technology
Most of the downsides of recombinant DNA technology are ethical in nature. Some people
feel that recombinant DNA technology goes against the laws of nature, or against their religious
beliefs, due to how much control this technology gives humans over the most basic buildings
blocks of life.
Other ethical concerns also exist. Some people worry that if companies can pay scientists
to patent, buy and sell genetic material, then genetic material could become an expensive
commodity. Such a system might lead to people having their genetic information stolen and used
without permission. It may sound odd, but such cases have already happened. In 1951, a scientist
used unique cells stolen from a woman named Henrietta Lacks to create an important cell line (the
HeLa cell line) which is still used in medical research today. Her family did not know about her
involuntary donation until after her death, and never received compensation, but others have
profited from the use of HeLa cells.
Many people worry about the safety of modifying food and medicines using recombinant
DNA technology. Although genetically modified foods seem safe in multiple studies, it is easy to
see why such fears exist.
What might happen if a crop of tomatoes with modified jellyfish genes to make them more
robust became common? What would happen to an unsuspecting person, who is allergic to
jellyfish, after eating one of these tomatoes? Would the person have a reaction? Some people fear
that such questions will not come up until it is too late.
Other people worry that humans may begin tampering too much with their own genetic
material and create societal problems. What if people use recombinant DNA technology to live

longer, become stronger or handpick certain traits for their offspring? Will societal division swell
between genetically modified people and "normal" people? These are questions that scientists and
the public will likely continue to consider as humanity moves toward a future where manipulating
DNA is easier than ever before.

2. Restriction enzymes
One of the most important steps in molecular biology, especially molecular genetics and
analysis, is the isolation of DNA from the human genome and make many copies of it. A restriction
enzyme is a kind of nuclease enzyme which is capable of cleaving double-stranded DNA. The
enzymes may cleave DNA at random or specific sequences which are referred to as restriction
sites (Fig 1). The recognition sites are palindromic in origin, that is, they are the sequences which
are read the same forward and backward. These restriction enzymes are produced naturally by
bacteria. The bacterial species use it as a form of defense mechanism against viruses. However, in
bacteria, restriction enzymes are present as a part of a combined system called the restriction
modification system. The bacterial species modify their own DNA with the help of enzymes which

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methylate it. This particular process of methylation of bacterial DNA protects it from cleavage
from its own restriction endonucleases.

Fig 1: The Restriction enzymes cleave DNA at random or specific sequences

Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them
for use). Because they cut within the molecule, they are often called restriction endonucleases.
A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides.
For example, the bacterium Hemophilus aegypticus produces an enzyme named Haelll that cuts
DNA wherever it encounters the sequence

5’GGCC3’
3’CCGG5’
The cut is made between the adjacent G and C. This particular sequence occurs at 11 places
in the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the enzyme
produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can
be separated from one another and the sequence of each determined. Haelll and Alul cut straight
across the double helix producing “blunt” ends. However, many restriction enzymes cut in an
offset fashion.
The ends of the cut have an overhanging piece of single-stranded DNA. These are called
“sticky ends” because they are able to form base pairs with any DNA molecule that contains the

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complementary sticky end (Fig.2). Any other source of DNA treated with the same enzyme will
produce such molecules. Mixed together, these molecules can join with each other by the base
pairing between their sticky ends. The union can be made permanent by another enzyme, DNA
ligase that forms covalent bonds along the backbone of each strand. The result is a molecule of
recombinant DNA (rDNA). Because recognition sequences and cleavage sites differ between
restriction enzymes, the length and the exact sequence of a sticky-end “overhang”, as well as
whether it is the 5′ end or the 3′ end strand that overhangs, depends on which enzyme produced it.
Base-pairing between overhangs with complementary sequences enables two fragments to be
joined or “spliced” by a DNA ligase.

Fig.2 Alu1 and HaeIII produce blunt ends. BamH1, HindIII and EcoR1 produce “sticky”
ends
A sticky-end fragment can be ligated not only to the fragment from which it was originally
cleaved, but also to any other fragment with a compatible sticky end. The sticky end is also
called a cohesive end or complementary end in some reference.
If a restriction enzyme has a non- degenerate palindromic (the sequence on one strand reads the
same in the same direction on the complementary strand e.g. GTAATG is not a palindromic
DNA sequence, but GTATAC is, GTATAC is complementary to CATATG) cleavage site, all
ends that it produces are compatible. Ends produced by different enzymes may also be
compatible.
Patterns of DNA Cutting by Restriction Enzymes:
(i) 5′ overhangs:
The enzyme cuts asymmetrically within the recognition site such that a short single-
stranded segment extends from the 5′ ends. BamHI cuts in this manner.

(ii) 3′ overhangs:

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 Again, we see asymmetrical cutting within the recognition site, but the result is a single-
stranded overhang from the two 3′ ends. Kpnl cuts in this manner.

Table: 1 Restriction ennzymes their sources and recognition sites.

(iii) Blunts:
Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends
without overhangs. Smal is an example of an enzyme that generates blunt ends.

The 5′ or 3′ overhangs generated by enzymes that cut asymmetrically are called sticky ends or
cohesive ends, because they will readily stick or anneal with their partner by base pairing.

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Types
There are two different kinds of restriction enzymes:
1. Exonucleases: restriction exonucleases are primarily responsible for hydrolysis of the
terminal nucleotides from the end of DNA or RNA molecule either from 5’ to 3’ direction
or 3’ to 5’ direction; for example- exonuclease I, exonuclease II, etc.
2. Endonuclease: restriction endonucleases recognize particular base sequences (restriction
sites) within DNA or RNA molecule and catalyze the cleavage of internal phosphodiester
bond; for exEcoRI, Hind III, BamHI, etc.
The first restriction enzyme to be discovered was Hind II in the year 1970. In 1978, Daniel
Nathans, Werner Arber, and Hamilton O. Smith were awarded the Nobel Prize for Physiology or
Medicine.
Restriction Enzyme Nomenclature
The very name of the restriction enzymes consists of three parts:
1. An abbreviation of the genus and the species of the organism to 3 letters, for example- Eco
for Escherichia coli identified by the first letter, E, of the genus and the first two letters, co,
of the species.
2. It is followed by a letter, number or combination of both of them to signify the strain of the
species.
3. A Roman numeral to indicate the order in which the different restriction-modification
systems were found in the same organism or strain per se.

Classification of Restriction Endonucleases

Based on the types of sequences identified, the nature of cuts made in the DNA, and the enzyme
structure, there are three classes:

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 1. Type I restriction enzymes,
2. Type II restriction enzymes, and
3. Type III restriction enzymes.
Type I Restriction Enzymes
• Type I restriction enzymes possess both restriction and modification activities. In this case,
the restriction will depend upon the methylation status of the target DNA sequence.
• Cleavage takes place nearly 1000 base pairs away from the restriction site. The structure
of the recognition site is asymmetrical. It is composed of 2 parts. One part of the recognition
site is composed of 3-4 nucleotides while the other one contains 4-5 nucleotides. The two
parts are separated by a non-specific spacer of about 6-8 nucleotides.
• For their function, the type I restriction enzymes require S- adenosylmethionine
(SAM), ATP, and Mg2+ .
• They are composed of 3 subunits, a specificity subunit which determines the recognition
site, a restriction subunit, and a modification subunit.
Type II Restriction Enzymes
• Two separate enzymes mediate restriction and modification. Henceforth, DNA can be
cleaved in the absence of modifying enzymes. Although the target sequence identified by
the two enzymes is the same, they can be separately purified from each other.
• The nucleotides are cleaved at the restriction site only. The recognition sequence is
rotationally symmetrical, called palindromic sequence. The specific palindromic site can
either be continuous (e.g., KpnI identifies the sequence 5´-GGTACC-3´) or non-
continuous (e.g., BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any
nucleotide).
• These require Mg2+ as a cofactor but not ATP.
• They are required in genetic mapping and reconstruction of the DNA in vitro only because
they identify particular sites and cleave at those sites only.
• The type II restriction enzymes first establish non-specific contact with DNA and bind to
them in the form of dimmers.
• The target sequence is then detected by a combination of two processes. Either the enzyme
diffuses linearly/ slides along the DNA sequence over short distances or hops/ jumps over
long distances.
• Once the target sequence is located, various conformational changes occur in the enzyme
as well as the DNA. These conformational changes, in turn, activate catalytic center.
• The phosphodiester bond is hydrolyzed, and the product is released.

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 Fig 3: Structures of free, nonspecific, and specific DNA-bound forms of BamHI

Type III Restriction Enzyme

• The type III enzymes recognize and methylate the same DNA sequence. However, they
cleave nearly 24-26 base pairs away.
• They are composed of two different subunits. The recognition and modification of DNA
are carried out by the first subunit- ‘M’ and the nuclease activity is rendered by the other
subunit ‘R’.
• DNA cleavage is aided by ATP as well as Mg2+ whereas SAM is responsible for
stimulating cleavage.

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 • Only one of the DNA strand is cleaved. However, to break the double-stranded DNA, two
recognition sites in opposite directions are required.
Table 2: Types of Restriction enzymes

Applications:

In various applications related to genetic engineering DNA is cleaved by using these

restriction enzymes.
• They are used in the process of insertion of genes into plasmid vectors during gene cloning
and protein expression experiments.

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 • Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing
single base changes in DNA known as single nucleotide polymorphisms (SNPs). This is
only possible if a mutation alters the restrictionsite present in the allele.
• Restriction enzymes are used for Restriction Fragment Length Polymorphism (RFLP)
analysis for identifying individuals or strains of a particular species.

Polynucleotide phosphorylase:
• Polynucleotide phosphorylase was first discovered from extracts of Azotobacter agile by
Grunberg-Manago and Ochoa.
• Polynucleotide phosphorylase (PNPase) catalyzes the synthesis of long chain
polyribonucleotides (RNA) in 5’ to 3’ direction from nucleotide diphosphates as precursors
and reversibly catalyzes phosphorolytic cleavage of polyribonucleotides in 3’ to 5’
direction with a release of orthophosphate in presence of inorganic phosphate.
• PNPase is a bifunctional enzyme and functions in mRNA processing and degradation
inside the cell.
• Structural and physiochemical studies in enzymes showed that it is formed of subunits. The
arrangements of the subunits may vary from species to species which would alter their
properties.
• These enzyme can catalyze not only the synthesis of RNA from the mixtures of naturally
occurring ribonucleoside diphosphates, but also that of non-naturally occurring
polyribonucleotides

Mechanism of action:
As mentioned earlier, polynucleotide phosphorylase is a bifunctional enzyme. The
mechanism of action of this enzyme can be represented by following reactions:

In E.coli, polynucleotide phosphorylase regulates mRNA processing either by adding

ribonucleotides to the 3’ end or by cleaving bases in 3’ to 5’ direction. The function of PNPase
depends upon inorganic phosphate (Pi) concentration inside the cell. The transcripts are
polyadenylated using enzyme polyadenylate polymerase I (PAPI). After primary
polyadenylylation of the transcript by PAP I, PNPase may bind to the 3′ end of the poly(A) tail.
PNPase works either degradatively or biosynthetically inside the cell depending on the Pi
concentration. Under high Pi concentration, it degrades the poly(A) tail releasing adenine
diphosphates. If the Pi concentration is low, PAP I initiates addition of one or more nucleotides to
the existing poly (A) tail and in the process generates inorganic phosphate. On dissociation of
PNPase, the 3′ end again is available to PAP I for further polymerization.

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Fig 4: Schematic representation of the role of PNPase in poly(A) tail metabolism in E. coli.

Function:
Different functions of Polynucleotide phosphorylase are:
• It is involved in mRNA processing and degradation in bacteria, plants, and in humans.
• It synthesizes long, highly heteropolymeric tails in vivo as well as accounts for all of the
observed residual polyadenylation in poly(A) polymerase I deficient strains.
• PNPase function as a part of RNA degradosome in E.coli cell. RNA degradosome is a
multicomponent enzyme complex that includes RNaseE (endoribinuclease),
polynucleotide phosphorylase (3’ to 5’ exonuclease), RhlB helicase (a DEAD box helicase)
and a glycolytic enzyme enolase. This complex catalyzes 3’ to 5’ exonuclease activity in
presence of ATP. In eukaryotes, the exosomes are located in nucleus and cytoplasm.
Degradsomes in bacteria and exosomes in eukaryotes are associated with processing,
control and turnover of RNA transcripts.
• In rDNA cloning technology, it has been used to synthesize radiolabelled
polyribonucleotides from nucleoside diphosphate monomers.

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Deoxyribonuclease (DNaseI):
• A nuclease enzyme that can catalyze the hydrolytic cleavage of phosphodiester bonds in
the DNA backbone are known as deoxyribonuclease (DNase).
• Based on the position of action, these enzymes are broadly classified as
endodeoxyribonuclease (cleave DNA sequence internally) and exodeoxyribonuclease
(cleave the terminal nucleotides)
• Unlike restriction enzymes, DNase does not have any specific recognition/restriction site
and cleave DNA sequence at random locations.
• There is a wide variety of deoxyribonucleases known which have different substrate
specificities, chemical mechanisms, and biological functions. They are:
1. Deoxyribonuclease I (DNaseI):
An endonuclease which cleaves double-stranded DNA or single stranded DNA. The
cleavage preferentially occurs adjacent to pyrimidine (C or T) residues. The major products are 5'-
phosphorylated bi-, tri- and tetranucleotides. It requires divalent ions (Ca2+ and Mn2+/Mg2+) for its
activity and creates blunt ends or 1-2 overhang sequences.
DNaseI is the most widely used enzyme in cloning experiments to remove DNA
contamination from mRNA preparation (to be used for cDNA library preparation, northern
hybridization, RT-PCR etc). The mode of action of DNaseI varies according to the divalent cation
used.
In the presence of magnesium ions (Mg+2), DNaseI hydrolyzes each strand of duplex DNA
producing single stranded nicks in the DNA backbone, generating various random cleavages. On
the other hand, in the presence of manganese ions (Mn+2), DNaseI cleaves both strands of a double
stranded DNA at approximately the same site, producing blunt ended DNA fragments or with 1-2
base overhangs. The two major DNases found in metazoans are: deoxyribonuclease I and
deoxyribonuclease II.
Some of the common applications of DNase I in rDNA technology have been mentioned below:
• Eliminating DNA contamination (e.g. plasmid) from preparations of RNA.
• Analyzing the DNA-protein interactions via DNA footprinting.
• Nicking DNA prior to radio-labeling by nick translation.

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 Fig 5: Action of DNase I in the presence of Mg+2 and Mn+2 ions. (Arrowhead denoting
random site of cleavage in double stranded DNA by DNase I)

2. DeoxyribonucleaseII (DNaseII):
It is a non-specific endonuclease with optimal activity at acidic pH (4.5-5.5) and conserved
from human to C.elegans.. It does not require any divalent cation for its activity. DNaseII initially
introduces multiple single stranded nicks in DNA backbone and finally generates 3’ phosphate
groups by hydrolyzing phosphodiester linkages.
This enzyme releases 3’phosphate groups by hydrolyzing phosphodiester linkage and
creating nicks in the DNA backbone. DNaseII acts by generating multiple single stranded nicks
followed by production of acid soluble nucleotides and oligonucleotides. The catalytic site of the
enzyme contains three histidine residues which are essential for enzyme activity.
Some of the common applications of DNase II are as follows:
• DNA fragmentation
• Molecular weight marker
• Cell apoptosis assays etc.
3. Exonuclease III:
Exonuclease III is a globular enzyme which has 3’→5’ exonuclease activity in a double stranded
DNA. The template DNA should be double stranded and the enzyme does not cleave single
stranded DNA. The enzyme shows optimal activity with blunt ended sequences or sequences with
5’ overhang. Exonuclease III enzyme has a bound divalent cation which is essential for enzyme
activity. The mechanism of the enzyme can be affected by variation in temperature, monovalent

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ion concentration in the reaction buffer, and structure and concentration of 3’termini. The enzyme
shows optimal activity at 37°C at pH 8.0. Various application of exonuclease III in molecular
cloning experiments are:
• To generate template for DNA sequencing
• To generate substrate for DNA labeling experiments
• Directed mutagenesis
• DNA-protein interaction assays (to find blockage of exonuclease III activity byprotein-
DNA binding) etc.

4. Mung bean nuclease:

As the name suggest, this nuclease enzyme is isolated from mung bean sprouts (Vigna
radiata). Mung bean nuclease enzymes can degrade single stranded DNA as well RNA. Under high
enzyme concentration, they can degrade double stranded DNA, RNA or even DNA/RNA hybrids.
Mung bean nuclease can cleave single stranded DNA or RNA to produce 5’-phosphoryl mono and
oligonucleotides. It requires Zn2+ ion for its activity and shows optimal activity at 37°C. The
enzyme works in low salt concentration (25mM ammonium acetate) and acidic pH (pH 5.0).
Treatment with EDTA or SDS results in irreversible inactivation of the enzyme. Mung bean
nuclease is less robust than S1 nuclease and easier to handle. It has been used to create blunt end
DNA by cleaving protruding ends from 5’ ends. This enzyme cannot produce nicks in a double
stranded DNA but at higher concentration, it can generate nicks and cleave double stranded DNA.
5. Phosphatase:
• Phosphatase catalyses the cleavage of a phosphate (PO4 -2 ) group from substrate by using
a water molecule (hydrolytic cleavage).
• This reaction is not reversible.
• This shows totally opposite activity from enzyme like kinase and phosphorylase that add a
phosphate group to their substrate. On the basis of their activity there are two types of
phosphatase i.e acid phosphatase and alkaline phosphatase. In both forms the alkaline
phosphatase are most common.
• Special class of phosphatase that remove a phosphate group from protein, called
“Phosphoprotein phosphatase”.

i. Acid phosphatase:
It shows its optimal activity at pH between 3 and 6, e.g. a lysosomal enzyme that
hydrolyze organic phosphates liberating one or more phosphate groups. They are
found in prostatic epithelial cells, erythrocyte, prostatic tissue, spleen, kidney etc.
ii. Alkaline phosphatase:
• Homodimeric enzyme which catalyzes reactions like hydrolysis and
transphosphophorylation of phosphate monoester.

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 • They show their optimal activity at pH of about 10.
• Alkaline phosphatase was the first zinc enzyme discovered having three closed
spaced metal ion. Two Zn+2 ions and one Mg+2 ion, in which Zn+2 ions are
bridges by Asp 51. The mechanism of action is based on reaction where a covalent
serine – phosphate intermediate is formed to produce inorganic phosphate and an
alcohol.
• In human body it is present in four isoforms, in which three are tissue specific
isoform i.e. placental, germ cell, intestinal and one is non tissue specific isoform.
The genes that encode for tissue specific isoforms are present on chromosome -2
p37-q37, while the genes for one non tissue specific are present on chromosome -
1 p34- p36.1.
• During post-translational modification, alkaline phosphatase is modified by
Nglycosylation. It undergoes a modification through which uptake of two Zn+2 ion
and one Mg+2 ion occurs which is important in forming active site of that enzyme.
Alkaline phosphatases are isolated from various sources like microorganisms,
tissue of different organs, connective tissue of invertebrate and vertebrate, and
human body.

Fig6: Action of alkaline phosphatase

• Bacterial alkaline phosphatase (BAP) - Bacterial alkaline phosphate is a
phosphomonoester that hydrolyzes 3’ and 5’ phosphate from nucleic acid (DNA/
RNA). It more suitably removes phosphate group before end labeling and remove
phosphate from vector prior to insert ligation. BAP generally shows optimum
activity at temperature 65°C. BAP is sensitive to inorganic phosphate so in presence
of inorganic phosphates activity may reduce.
• Calf intestinal alkaline phosphatase (CIP) – It is isolated from calf intestine,
which catalyzes the removal of phosphate group from 5’ end of DNA as well as
RNA. This enzyme is highly used in gene cloning experiments, as to make a
construct that could not undergo self-ligation. Hence after the treatment with CIP,
without having a phosphate group at 5’ ends a vector cannot self ligate and
recircularise. This step improves the efficiency of vector containing desired insert.
• Shrimp alkaline phosphatase (SAP) - Shrimp alkaline phosphatase is highly
specific, heat labile phosphatase enzyme isolated from arctic shrimp (Pandalus
borealis). It removes 5’ phosphate group from DNA, RNA, dNTPs and proteins.
SAP has similar specificity as CIP but unlike CIP, it can be irreversibly inactivated
by heat treatment at 65°C for 15mins. SAP is used for 5’ dephosphorylation during
cloning experiments for various application as follows:
a. Dephosphorylate 5’-phosphate group of DNA/RNA for subsequent
b. labeling of the ends.
c. linearized plasmid.

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 d. To prepare PCR product for sequencing.
e. To inactivate remaining dNTPs from PCR product (for downstream
sequencing appication).
Two primary uses for alkaline phosphatase in DNA modification:
Removing 5’ phosphate from different vector like plasmid, bacteriophage after treating
with restriction enzyme. This treatment prevents self ligation because unavailability of phosphate
group at end. So, this treatment greatly enhances the ligation of desired insert. During ligation of
desired insert, the complementary ends of the insert and vector will come to proximity of each
other (only for sticky ends but not for blunt ends). One strand of the insert having 5’-phosphate
will ligate with the 3’OH of the vector and the remaining strand will have a nick. This nick will be
sealed in the next step by ligase enzyme in the presence of ATP. It is used to remove 5’ phosphate
from fragment of DNA prior to labeling with radioactive phosphate.
6. Methylase:
• Methyltransferase or methylase catalyzes the transfer of methyl group (-CH3) to its
substrate. The process of transfer of methyl group to its substrate is called
methylation.
• Methylation is a common phenomenon in DNA and protein structure.
• Methyltransferase uses a reactive methyl group that is bound to sulfur in Sadenosyl
methionine (SAM) which acts as the methyl donor.
• Methylation normally occurs on cytosine (C) residue in DNA sequence. In protein,
methylation occurs on nitrogen atom either on N-terminus or on the side chain of
protein.
• DNA methylation regulates gene or silence gene without changing DNA
sequences, as a part of epigenetic regulation.
• In bacterial system, methylation plays a major role in preventing their genome from
degradation by restriction enzymes. It is a part of restriction – modification system
in bacteria.
• In bacterial system, methylation plays a major role in preventing their genome
Methyltransferase can be classified in three groups:
a) m6A-generates N6 methyladenosine,
b) m4C-generates N4 methylcytosine,
c) m5C-generatesN5 methylcytosine.

m6A and m4C methyltransferase are primarily found in prokaryotes. These enzymes are
responsible for methylation of DNA sequences in order to prevent the host from digesting its own
genome via its restriction enzyme.

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 Fig 7: Activity of restriction and methylase enzymes
Restriction enzyme EcoRI cleaves within the recognition sequence if the DNA is
unmethylated. On methylation by methylases, the restriction enzyme EcoRI is inhibited from
cleaving within the restriction site.
Some common examples of methytransferases are: DNA adenyl methytransferase (DAM),
histone methyltransferase, O-methyltransferase etc. DAM methylase is generally used in
recombinant DNA technology which can methylate adenine (A) in the sequence 5’GATC3’. This
enzyme can methylate a newly synthesized DNA strand on specific sites.

7. Ligases:
• DNA ligase catalyses the formation of phosphodiester bond between two
deoxynucleotide residues of two DNA strands.
• DNA ligase enzyme requires a free hydroxyl group at the 3´ -end of one DNA chain
and a phosphate group at the 5´-end of the other and requires energy in the process.
• E.coli and other bacterial DNA ligase utilizes NAD+ as energy donor, whereas in
T4 bacteriophage, T4 DNA ligase uses ATP as cofactor.

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• The role of DNA ligase is to seal nicks in the backbone of double-stranded DNA
after lagging strand formation to join the okazaki fragments.
• This joining process is essential for the normal synthesis of DNA and for repairing
damaged DNA. It has been exploited by genetic engineers to join DNA chains to
form recombinant DNA molecules. Usually single stranded break are repaired
using the complimentary strand as the template but sometimes double stranded
breaks can also be repaired with the help of DNA ligase IV.
• The most widely used DNA ligase is isolated from T4 bacteriophage. T4 DNA
ligase needs ATP as a cofactor. The enzyme from E. coli uses cofactor NAD.
Except this, the catalysis mechanism is somewhat similar for both the ligases. The
role of cofactor is splitting and forming an enzyme-AMP complex which further
aids in formation of phosphodiester bonds between hydroxyl and phosphate groups
by exposing them.
i. Mechanism of Action of DNA Ligases:
• ATP, or NAD+, reacts with the ligase enzyme to form a covalent enzyme–AMP
complex in which the AMP is linked to ε-amino group of a lysine residue in the
active site of the enzyme through a phosphoamide bond.
• The AMP moiety activates the phosphate group at the 5´-end of the DNA molecule
to be joined. It is called as the donor.
• The final step is a nucleophilic attack by the 3´-hydroxyl group on this activated
phosphorus atom which acts as the acceptor. A phosphodiester bond is formed and
AMP is released.
• The reaction is driven by the hydrolysis of the pyrophosphate released during the
formation of the enzyme–adenylate complex. Two high-energy phosphate bonds
are spent in forming a phosphodiester bond in the DNA backbone with ATP serving
as energy source.
• The temperature optimum for T4 DNA ligase mediated ligation in vitro is 16˚C.
However ligation is also achieved by incubation at 4˚C by incubating over night or
at room temperature condition by incubating for 30 minutes.
• Adenylate and DNA-adenylate are the important intermediates of the
phosphodiester bond forming pathway.

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 Fig 8: The mechanism of DNA joining by DNA ligase

ii. Application:
• DNA ligase enzyme is used by cells to join the “okazaki fragments” during DNA
replication process. In molecular cloning, ligase enzyme has been routinely used to
construct a recombinant DNA. Followings are some of the examples of application
of ligase enzyme in molecular cloning.Joining of adapters and linkers to blunt end
DNA molecule.
• Cloning of restricted DNA to vector to construct recombinant vector.

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 Fig 9: Ligation of a gene fragment into the vector and transformation of the
cell
8. Polynucleotide Kinase:
• PNK is a homotetramer with phosphatase activity at 3’ end and kinase activity at
5’ end with a tunnel like active site. The active site has side chains which interact
with NTP donor’s beta-phosphate and 3’ phosphate of acceptor with an acid which
activated 5’ –OH. Lys-15 and Ser-16 are important for the kinase activity of the
enzyme.
• The basic residues of active site of PNK interact with the negatively charged
phosphates of the DNA.
• Polynucleotide kinase (PNK) catalyzes the transfer of a phosphate group (PO4 -2 )
from γ position of ATP to the 5' end of either DNA or RNA and nucleoside
monophosphate.
• PNK can convert 3' PO4/5' OH ends into 3' PO4/5' PO4 ends which blocks further
ligation by ligase enzyme.
• PNK is used to label the ends of DNA or RNA with radioactive phosphate group.
• T4 polynucleotide kinase is the most widely used PNK in molecular cloning
experiments, which was isolated from T4 bacteriophage infected E.coli.
PNK carries out two types of enzymatic activity:
• Forward reaction: γ-phosphate is transferred from ATP to the 5' end of a
polynucleotide (DNA or RNA). 5’ phosphate is not present either due to chemical
synthesis or dephosphorylation. The 5’ OH nucleophile is activated by abstraction
of the proton. Asp35 of PNK forms the co-ordinate bond with 5’ OH and attacks γ
phosphorus forming an intermediate.
• Exchange reaction: target DNA or RNA having a 5' phosphate is incubated with
an excess of ADP - where PNK transfers the phosphate from the nucleic acid to an
ADP, forming ATP. PNK then performs a forward reaction and transfer a
phosphate from ATP to the target nucleic acid. Exchange reaction is used to label
with radioactive phosphate group.

21
Fig 10: Polynucleotide kinase reaction (A) forward (B) exchange

9. Ribonuclease (RNase):
• Nuclease that can catalyze hydrolysis of ribonucleotides from either single stranded
or double stranded RNA sequence are called ribonucleotides (RNase).
• RNase are classified into two types depending on position of cleavage, i.e.
endoribonuclease (cleave internal bond) and exoribonuclease (cleave terminal
bond).
• RNase is important for RNA maturation and processing.
• RNaseA and RNaseH play important role in initial defence mechanism against
RNA viral infection. Two common types of ribonucleases are discussed below:
9.1. RibonucleaseA (RNaseA):
• An endo-ribonuclease that cleaves specifically single-stranded RNA at the 3' end
of pyrimidine residues.
• The RNA is degraded into 3'-phosphorylated mononucleotides C and U residues
and oligonucleotides in the form of 2', 3'-cyclic monophosphate intermediates.
• Optimal temperature for RNaseA is 60˚C (activity range 15-70˚C) and optimal pH
is 7.6.
• RNaseA has two histidine residues in its active site (His12 and His119). In the first
step, His12 acts as a base; accepting proton forming a nucleophile which then
attacks positively charged phosphorus atom. His119 acts as an acid in this case,
donating a proton to oxygenated P-O-R’ bond. The imidazole side chain acts as
base in His 12 here.
• The side chain of Lys41 and Phe120 further stabilize the transition state. Nitrogen
of the main chain of Phe120 donates hydrogen, thus bonding with the unbound
oxygen atom.
• In the second step the acid base activities get reversed and His119 accepts proton
from water causing hydroxyl attack on cyclic intermediate.

22
 • Activity of RNaseA can be inhibited by alkylation of His12 and His119 residue
essential for activity of the enzyme.

Fig 11: Mechanism of action of RNase A

Application:
• It is used to remove RNA contamination from DNA sample.

9.2 RibonucleaseH:
• Non-specific endoribonuclease that degrades RNA by hydrolytic mechanism from
DNA/RNA duplex resulting in single stranded DNA.
• Enzyme bound divalent metal ion is a cofactor here. The product formed is 5’
phosphorylated ssDNA.
• During cDNA library preparation from RNA sample, RNaseH enzyme is used to
cleave RNA strand of DNA-RNA duplex.

23
Fig 12: Schematic representation of cDNA preparation from mRNA

24
 SCHOOL OF BIO AND CHEMICAL ENGINEERING
DEPARTMENT OF BIOTECHNOLOGY

UNIT – 2– GENETIC ENGINEERING – SBB2102

1
 BIOLOGY OF CLONING VECTORS

1. Plasmids
A plasmid is a small, circular piece of DNA that is different than the chromosomal DNA,
which is all the genetic material found in an organism’s chromosomes. It replicates independently
of chromosomal DNA. Plasmids are mainly found in bacteria, but they can also be found in archaea
and multicellular organisms. Plasmids usually carry at least one gene, and many of the genes that
plasmids carry are beneficial to their host organisms. Although they have separate genes from their
hosts, they are not considered to be independent life.
In addition to bacterial chromosome (nucleoid), bacterial cells normally contain genetic
elements in their cytoplasm. These genetic elements exist and replicate separately from the
chromosome and are called plasmids. The very existence of plasmids in bacterial cytoplasm was
revealed by Lederberg in 1952 while working on conjugation process in bacteria.
Lederberg coined the term ‘plasmid’ to refer to the transmissible genetic elements that were
transferred from one bacterial cell to another and determined the maleness in bacteria. Literally,
thousands of plasmids are now known; over 300 different naturally occurring plasmids have been
isolated from strains of Escherichia coli alone. Besides naturally occurring plasmids, many
artificially modified plasmids have been developed and used as vectors in the process of gene
cloning (genetic engineering).
2.1. Physical Nature and Copy Number of Plasmids
The physical nature of plasmids is quite simple. They are small double-stranded DNA
molecules. Majority of the plasmids are circular, but many linear plasmids are also known.
Naturally occurring plasmids vary in size from approximately 1 kilobase to more than 1 megabase,
and a typical plasmid DNA is considered to be less than 5% the size of the bacterial chromosome.
Most of the plasmid DNA isolated from bacterial cells exist in the supercoil configuration, which
is the most compact form for DNA to exist within the cell.
The copy number refers to the fact that different plasmids occur in cells in different
numbers. Some plasmids are present in the cell in only 1-3 copies, whereas others may be present
in over 100 copies. Copy number is controlled by genes on the plasmid and by interactions between
the host and the plasmid.
2.2. Properties of Plasmids:
1. They are specific to one or a few particular bacteria.
2. They replicate independently of the bacterial chromosome.
3. They code for their own transfer.
4. They act as episomes and reversibly integrate into bacterial chromosome.
5. They may pick-up and transfer certain genes of bacterial chromosome,
6. They may affect certain characteristics of the bacterial cell,
7. Plasmids differ from viruses in following two ways.

2
 8. They do not cause damage to cells and generally are beneficial.
9. They do not have extracellular forms and exist inside cells simply as free and typically
circular DNA.
2.3. Incompatibility of Plasmids:
In some cases, a single bacterial cell contains several different types of plasmids. Borrelia
burgdorferi that causes Lyme disease, for convenience, possesses 17 different circular and linear
plasmids. In a condition when a plasmid is transferred to a new bacterial cell that already possesses
another plasmid, it is commonly observed that the second (transferred) plasmid is not
accommodated and is lost during subsequent replication. This condition is called plasmid
incompatibility and the two plasmids are said to be incompatible. A number of incompatibility
groups of plasmids have been recognised in bacteria. The plasmids of one incompatibility group
exclude each other from replicating in the cell but generally coexist with plasmids from other
groups.
Plasmids of an incompatibility group share a common mechanism of regulating their
replication and are thus related’ to one another. Therefore, although a bacterial cell may possess
various types of plasmids, each is genetically distinct.
2.4. Types of Plasmids:
Various types of plasmids naturally occur in bacterial cells, and the most favoured
classification of such plasmids is based on their main functions encoded by their own genes.
Following are the main type of plasmids recognised on the basis of above mentioned
characteristic feature:
2.4.1. F-plasmid (or F-factor):
F-plasmid or F-factor (“F” stands for fertility) is the very well characterised plasmid. It
plays a major role in conjugation in bacteria E. coli and was the first to be described. It is this
plasmid that confers ‘maleness’ on the bacterial cells; the term ‘sex-factor’ is also used to refer to
F-plasmid because of its this property. F-plasmid is a circular dsDNA molecule of 99,159 base
pairs.
The genetic map of the F-plasmid is shown in Fig. 5.31. One region of the plasmid contains
genes involved in regulation of the DNA replication (rep genes), the other region contains
transposable elements (IS3, Tn 1000, IS3 and IS2 genes) involved in its ability to function as an
episome, and the third large region, the tra region, consists of tra genes and possesses ability to
promote transfer of plasmids during conjugation. Example F-plasmid of E. coli.

3
 Fig 1: Genetic map of the F plasmid of E.coli and Resistance plasmid R100

2.4.2. R-plasmids:
R-plasmids are the most widespread and well-studied group of plasmids conferring resistance
(hence called resistant plasmids) to antibiotics and various other growth inhibitors. R- plasmids
typically have genes that code for enzymes able to destroy and modify antibiotics. They are not
usually integrated into the host chromosome. Some R-plasmids possess only a single resistant gene
whereas others can have as many as eight.
Plasmid R 100, for example, is a 94.3 kilobase-pair plasmid (Fig. 5.32) that carries resistant
genes for sulfonamides, streptomycin and spectinomycin, chloramphenicol, tetracyclin etc. It also
carries genes conferring resistance to mercury. Many R-plasmids are conjugative and possess
drug- resistant genes as transposable elements, they play an important role in medical
microbiology as their spread through natural populations can have profound consequences in the
treatment of bacterial infections.
2.4.3. Virulence-plasmids:
Virulence-plasmids confer pathogenesity on the host bacterium. They make the bacterium more
pathogenic as the bacterium is better able to resist host defence or to produce toxins. For example,
Ti-plasmids of Agrobacterium tumefaciens induce crown gall disease of angiospermic plants;
entertoxigenic strains of E. coli cause traveller’s diarrhoea because of a plasmid that codes for an
enterotoxin which induces extensive secretion of water and salts into the bowel.

4
2.4.4. Col-plasmids:
Col-plasmids carry genes that confer ability to the host bacterium to kill other bacteria by secreting
bacteriocins, a type of proteins. Bacteriocins often kill cells by creating channels in the plasma
membrane thus increasing its permeability. They also may degrade DNA or RNA or attack
peptidoglycan and weaken the cell-wall. Bacteriocins act only against closely related strains. Col
E1 plasmid of E. coli code for the synthesis of bacterioein called colicins which kill other
susceptible strains of E. coli. Col plasmids of some E.coli code for the synthesis of bacteriocin,
namely cloacins that kill Enterobacter species. Lactic acid bacteria produce bacteriocin NisinA
which strongly inhibits the growth of a wide variety of gram-positive bacteria and is used as a
preservative in the food industry.
2.4.5. Metabolic plasmids:
Metabolic plasmids (also called degradative plasmids) possess genes to code enzymes that
degrade unusual substances such as toluene (aromatic compounds), pesticides (2, 4-dichloro-
phenoxyacetic acid), and sugars (lactose).
TOL (= pWWO) plasmid of Pseudomonas putida is an example. However, some metabolic
plasmids occurring in certain strains of Rhizobium induce nodule formation in legumes and carry
out fixation of atmospheric nitrogen.

Fig 2: Interconversion of relaxed and supercoiled DNA in bacteria

5
2.5. Replication of Plasmids:
Plasmids replicate autonomously because they have their own replication origins. The
enzymes involved in plasmid replication are normal cell enzymes particularly in case of small
plasmids. But, some large plasmids carry genes that code for enzymes that are specific for plasmid
replication.
Plasmids possess relatively few genes, generally less than 30, and the genes are concerned
primarily with control of the replication initiation process and with apportionment of the replicated
plasmids between daughter cells; the genetic information carried in plasmid genes is not essential
to the host because the bacteria that lack them usually function normally.
Since the plasmid DNA is of small size, the whole process of its replication takes place very
quickly, perhaps in 1/10 or less of the total time of cell division cycle.
Most plasmids in gram-negative bacteria replicate in a manner similar to the replication of bacterial
chromosome involving initiation at the replication origin site and bidirectional replication around
the DNA circle giving a theta (Ө) intermediate.
However, some plasmids of gram-negative bacteria replicate by unidirectional method. Most
plasmids of gram-positive bacteria replicate by a rolling circle mechanism similar to that used by
phage φx174. Most linear plasmids replicate by means of a mechanism that involves a protein
bound to the 5′-end of each DNA strand that is used in priming DNA synthesis.
2.6. Plasmid Curing:
Plasmids can be eliminated from bacterial cells, and this process is called curing. Curing
may take place spontaneously or it may be induced by various treatments, which inhibit plasmid
replication but do not affect bacterial chromosome replication and cell reproduction. The inhibited
plasmids are slowly diluted out of the growing bacterial population.
Some commonly used curing treatment agents are acridine dyes, ultraviolet (UV) and
ionizing radiation, thymine starvation and growth above optimal temperatures. These curing
treatment agents interfere with plasmid replication than with bacterial chromosome replication.
2.7 Use of Plasmids as Cloning Vectors:
Significance of plasmids dramatically increased with the advent of recombinant DNA
technology as they became the first cloning vectors, and even today they are the most widely used
cloning vectors especially in gene cloning in bacteria.
They enjoy this status because they have very useful properties as cloning vectors that
include:
(i) Small size, which makes the plasmid easy to isolate and manipulate;
(ii) Independent origin of replication, which allows plasmid replication in the cell to proceed
independently from direct chromosomal control;
6
(iii) Multiple copy number, which makes them to be present in the cell in several copies so that
amplification of the plasmid DNA becomes easy; and
(iv) Presence of selectable markers such as antibiotic resistance genes, which make detection and
selection of plasmid-containing clones easier.
The plasmid vector is isolated from the bacterial cell and at one site by restriction enzyme. The
cleavage converts the circular plasmid DNA into a linear DNA molecule.
Now the two open ends of linear plasmid are joined to the ends of the foreign DNA to be
inserted with the help of enzyme DNA ligase. This regenerates a circular hybrid or chimeric
plasmid, which is transferred to a bacterium wherein it replicates and perpetuates indefinitely.
One of the most widely used plasmids in gene cloning in bacteria is pBR322, which has
both resistance genes for ampicillin and tetracycline and many restriction sites. When a foreign
DNA is inserted into the ampicillin resistance gene of pBR322, the plasmid is no longer able to
confer resistance to ampicillin.
2.8. Applications of Plasmids
Humans have developed many uses for plasmids and have created software to record the
DNA sequences of plasmids for use in many different techniques. Plasmids are used in genetic
engineering to amplify, or produce many copies of, certain genes. In molecular cloning, a plasmid
is a type of vector. A vector is a DNA sequence that can transport foreign genetic material from
one cell to another cell, where the genes can be further expressed and replicated. Plasmids are
useful in cloning short segments of DNA. Also, plasmids can be used to replicate proteins, such
as the protein that codes for insulin, in large amounts. Additionally, plasmids are being investigated
as a way to transfer genes into human cells as part of gene therapy. Cells may lack a specific
protein if the patient has a hereditary disorder involving a gene mutation. Inserting a plasmid into
DNA would allow cells to express a protein that they are lacking.
2.9. Origin of replication.
Required for automous replication of the plasmid using the host's replication machinery.
Almost all commonly used plasmids are based on the ColE1 origin of replication (ori). It is worth
noting that bacterial origins of replication are tightly regulated. While R factors are smaller than
the host genome (105 bp compared to 5x106 bp), replication of these factors to high copy number
in the host places a considerable load on the host replication machinery. Naturally occuring origins
of replication are therefore negatively regulated to keep copy number down (typically 5 to 10
copies per cell). While high copy number is disadvantageous in a natural system, it is a desirable
feature in a cloning vector - since the whole idea is to be able to easily isolate substantial quantities
of particular DNA sequences. Therefore considerable work has gone into engineering the ColE1
ori such that the negative regualtory mechanisms that limit episome copy number are disabled.
Modern plasmid vectors are therefore often called 'runaway replicons' and are present at 100 to
1000 copies per cell.

7
2.10 Selectable Markers
Selectable markers are essential for the identification of bacteria containing recombinant
plasmids. Selection can be divided into two types:
Positive Selection
Positive selection is used to identify bacteria that contain a plasmid. The most common markers
used for positive selection are the antibiotic resistance genes carried by the original R factors.
While many antibiotics and resistance genes are available, the commonly used ones fall into two
general classes:
Antibiotics affecting cell wall synthesis
Ampicillin is a beta-lactam based antibiotic that acts by inhibiting the synthesis of the
bacterial peptidoglycan cell wall. Sensitive bacteria are not actively 'killed', but on cell division
are unable to synthesize the cell wall and suffer from osmotic lysis.
The enzyme beta-lactamase is secreted into the periplasmic space where it breaks down the
antibiotic, allowing cell wall synthesis to proceed.
Antibiotics affecting translation:
The antibiotics tetracycline, kanamycin and chloramphenicol all act by inhibiting translation. The
covalent modification (phosphorylation, acetylation) of these antibiotics blocks their interaction
with the translation apparatus.
Positive selection is particularly important when introducing plasmids into bacteria by
transformation. At best, only about 1 in 10,000 bacteria picks up a plasmid that carries the
antibiotic resistance. A strong positive selection system is essential to eliminate the 9,999 bacteria
that didn't pick up a plasmid from the one that did. By plating the transformation products directly
on antibiotic plates, all untransformed bacteria die and only those containing the plasmid (and
antibiotic resistance marker) grow to form colonies.
Negative Selection
The ligation of foreign DNA fragments into plasmid vectors is a relatively inefficient
process - ligation can either produce recircularized plasmid with no insert, or plasmids containing
a foreign DNA insert. These products are then transformed into an antibiotic sensitive bacterial
host, an positive selection applied to identify bacteria that contain a plasmid. A second selection
system is necessary to distinguish between plasmids that merely recircularized from those that
carry a foreign DNA insert.
In order to identify those plasmids carrying a foreign DNA fragment, the site of insertion is chosen
such that insertion disrupts a selectable marker - a phenomenon we call insertional inactivation.
Two types of selectable markers are used for negative selection

8
Insertional inactivation of antibiotic resistance.
In order to use antibiotic resistance as a negative as well as a positive selection system, the
plasmid vector must carry two different antibiotic resistance genes. An example of such a vector
is pBR322.

• pBR322 carries both an ampicllin and a tetracycline resistance gene.

• The phenotype of bacteria containing the intact plasmid is Ampr Tetr
• Insertion of foreign DNA into
• the Pst I site located in the Ampr gene results in an Amps Tetr phenotype.
• Conversely, insertion of foreign DNA into the EcoRI, Hind III or Sal I sites located
in the Tetr gene results in an
• Ampr Tets phenotype.
• In order to reveal these phenotypes, transformants are first plated on positive
selective media Tet or Amp respectively).
• Positively selected colonies are then restreaked on positive selective media
(master plate from which we recover our desired vector + insert) and on the
negative selection media. (Amp or Tet media respectively)

9
Insertional Inactivation of Enzymatic Activity

While the insertional inactivation of antibiotic resistance works, it requires a lot of

manipulation - picking the positively selected bacteria and replating on negative selection media
etc. In addition to the tedium of picking colonies, vectors like pBR322 also suffer from a paucity
of convenient restriction sites at which to insert the foreign DNA fragemnts.

These limitations promted the development of a set of host-vector systems in which it is

possible to positively select for bacteria carrying a plasmid and simultaneously select for
insertional inactivation of enzymatic activity. This system is based on our old friend, the beta-
galactosidase gene of the E coli lac operon.

2.11. Examples of Cloning Vector:

a pBR322
• pBR322 is a widely-used E. coli cloning vector. It was created in 1977 in thelaboratory of
Herbert Boyer at the University of California San Francisco. The pstands for "plasmid" and
BR for "Bolivar" and "Rodriguez", researchers who constructed it.
• pBR322 is 4361 base pairs in length.
• pBR322 plasmid has the following elements
• rep” replicon from plasmid pMB1 which is responsible for replication of the plasmid.
• “rop” gene encoding Rop protein. Rop proteins are associated with stability of RNAI-
RNAII complex and also decrease copy number. The source of “rop” gene is
pMB1plasmid.
• “tet” gene encoding tetracycline resistance derived from pSC101 plasmid.
• “bla” gene encoding β lactamase which provide ampicillin resistance (source: transposon
Tn3).

10
 Fig 3: PlasmidPBR322

b. pUC plasmids:
• pUC plasmids are small, high copy number plasmids of size 2686bp.
• This series of cloning vectors were developed by Messing and co-workers in the University
of California. The p in its name stands for plasmid and UC represents the University of
California.
• pUC vectors contain a lacZ sequence and multiple cloning site (MCS) within lacZ. This
helps in use of broad spectrum of restriction endonucleases and permits rapid visual
detection of an insert.
• pUC18 and pUC19 vectors are identical apart from the fact that the MCS is arranged in
opposite orientation. pUC vectors consists of following elements:

✓ pMB1 “rep” replicon region derived from plasmid pBR322 with single point
mutation (to increase copy number).
✓ “bla” gene encoding β lactamase which provide ampicillin resistance which is
derived from pBR322. This site is different from pBR322 by two point mutations.
✓ E.coli lac operon system.
“rop” gene is removed from this vector which leads to an increase in copy number
An MCS is a short DNA sequence consisting of restriction sites for many different
restriction endonucleases. MCS escalates the number of potential cloning strategies available by
extending the range of enzymes that can be used to generate a restriction fragment suitable for
cloning. By combining them within a MCS, the sites are made contiguous, so that any two sites
within it can be cleaved simultaneously without excising vector sequences. The MCS is inserted
into the lacZ sequence, which encodes the promoter and the α-peptide of β-galactosidase. Insertion
of the MCS into the lacZ fragment does not affect the ability of the α-peptide to mediate
complementation, while cloning DNA fragments into the MCS does. Therefore, recombinants can
be detected by blue/white screening on growth medium containing X gal in presence of IPTG as
an inducer.

11
 Fig 4: pUC plasmid

2.12. Bacteriophage
Bacteriophage, also called phage or bacterial virus, any of a group of viruses that infect
bacteria. Bacteriophages were discovered independently by Frederick W. Twort in Great Britain
(1915) and Félix d’Hérelle in France (1917). D’Hérelle coined the term bacteriophage, meaning
“bacteria eater,” to describe the agent’s bacteriocidal ability. Bacteriophages also infect the single-
celled prokaryotic organisms known as archaea.
Characteristics of Bacteriophages
Thousands of varieties of phages exist, each of which may infect only one type or a few
types of bacteria or archaea. Phages are classified in a number of virus families; some examples
include Inoviridae, Microviridae, Rudiviridae, and Tectiviridae. Like all viruses, phages are simple
organisms that consist of a core of genetic material (nucleic acid) surrounded by a protein capsid.
The nucleic acid may be either DNA or RNA and may be double-stranded or single-stranded.
There are three basic structural forms of phage: an icosahedral (20-sided) head with a tail, an
icosahedral head without a tail, and a filamentous form.
All viruses depend on cells for reproduction and metabolic processes. By themselves,
viruses do not encode for all of the enzymes necessary for viral replication. But within a host cell,
a virus can commandeer cellular machinery to produce more viral particles. Bacteriophages
replicate only in the cytoplasm, since prokaryotic cells do not have a nucleus or organelles. In
eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed
in the large DNA viruses, such as the poxviruses, that can replicate in the cytoplasm. RNA viruses
that infect animal cells often replicate in the cytoplasm.

The Life Cycle of Viruses with Prokaryote Hosts

12
 The life cycle of bacteriophages has been a good model for understanding how viruses
affect the cells they infect, since similar processes have been observed for eukaryotic viruses,
which can cause immediate death of the cell or establish a latent or chronic infection. Virulent
phages typically lead to the death of the cell through cell lysis. Temperate phages, on the other
hand, can become part of a host chromosome and are replicated with the cell genome until such
time as they are induced to make newly assembled viruses, or progeny viruses.

The Lytic Cycle

During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new
phages, and destroys the cell. T-even phage is a good example of a well-characterized class of
virulent phages. There are five stages in the bacteriophage lytic cycle (see Figure 5). Attachment
is the first stage in the infection process in which the phage interacts with specific bacterial surface
receptors (e.g., lipopolysaccharides and OmpC protein on host surfaces). Most phages have a
narrow host range and may infect one species of bacteria or one strain within a species. This unique
recognition can be exploited for targeted treatment of bacterial infection by phage therapy or for
phage typing to identify unique bacterial subspecies or strains. The second stage of infection is
entry or penetration. This occurs through contraction of the tail sheath, which acts like a
hypodermic needle to inject the viral genome through the cell wall and membrane. The phage head
and remaining components remain outside the bacteria.
The third stage of infection is biosynthesis of new viral components. After entering the
host cell, the virus synthesizes virus-encoded endonucleases to degrade the bacterial chromosome.
It then hijacks the host cell to replicate, transcribe, and translate the necessary viral components
(capsomeres, sheath, base plates, tail fibers, and viral enzymes) for the assembly of new viruses.
Polymerase genes are usually expressed early in the cycle, while capsid and tail proteins are
expressed later. During the maturation phase, new virions are created. To liberate free phages, the
bacterial cell wall is disrupted by phage proteins such as holin or lysozyme. The final stage is
release. Mature viruses burst out of the host cell in a process called lysis and the progeny viruses
are liberated into the environment to infect new cells.

Figure 5. A virulent phage shows only the lytic cycle pictured here. In the lytic cycle, the phage
replicates and lyses the host cell.

13
The Lysogenic Cycle
In a lysogenic cycle, the phage genome also enters the cell through attachment and
penetration. A prime example of a phage with this type of life cycle is the lambda phage. During
the lysogenic cycle, instead of killing the host, the phage genome integrates into the bacterial
chromosome and becomes part of the host. The integrated phage genome is called a prophage. A
bacterial host with a prophage is called a lysogen. The process in which a bacterium is infected by
a temperate phage is called lysogeny. It is typical of temperate phages to be latent or inactive
within the cell. As the bacterium replicates its chromosome, it also replicates the phage’s DNA
and passes it on to new daughter cells during reproduction. The presence of the phage may alter
the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase
bacterial virulence). This change in the host phenotype is called lysogenic conversion or phage
conversion. Some bacteria, such as Vibrio cholerae and Clostridium botulinum, are less virulent
in the absence of the prophage. The phages infecting these bacteria carry the toxin genes in their
genome and enhance the virulence of the host when the toxin genes are expressed. In the case of
V. cholera, phage encoded toxin can cause severe diarrhea; in C. botulinum, the toxin can cause
paralysis. During lysogeny, the prophage will persist in the host chromosome until induction,
which results in the excision of the viral genome from the host chromosome. After induction has
occurred the temperate phage can proceed through a lytic cycle and then undergo lysogeny in a
newly infected cell (see Figure 6).

14
Figure 6. A temperate bacteriophage has both lytic and lysogenic cycles. In the lysogenic cycle,
phage DNA is incorporated into the host genome, forming a prophage, which is passed on to
subsequent generations of cells. Environmental stressors such as starvation or exposure to toxic
chemicals may cause the prophage to be excised and enter the lytic cycle.

Morphology of Phage Lambda:

Morphological structure of phage λ is given in Fig. 18.11A. The head has 20 faces. A 20-
faces 3-D picture is called an icosahedron. The head is made of protein of several types and
contains a 46,500 bp long genomic (g) DNA. The phage λ contains double stranded circular DNA
of about 17 µm in length packed in protein head of capsid. The head is 55 nm in diameter consisting
of 300-600 capsomers (subunits) of 37,500 Daltons.

The capsomers are arranged in clusters of 5 and 6 subunits i.e. pentamers and hexamers.
The head is joined to a non-contractile 180 µm long tail by a connector. The tail consists of 35
stacked discs. It ends in a fiber. There is a hole in capsid through which passes this narrow neck
portion expanding into a knob like structure inside. The tail possesses a thin tail fibre (25 nm long)
at its end which recognises the hosts. Also the tail consists of about 35 stacked discs or annuli.
Unlike T-even phage, it is a simple structure devoid of the tail sheath. Bacteriophage A falls under
the family Siphoviridae of the Group I (dsDNA viruses). Phage lambda is a virus of E. coli K12
which after entering inside host cell normally does not kill it in-spite of being capable of destroying
the host.
Therefore, it leads its life cycle in two different ways, one as virulent virus and the second
as non-virulent. The virulent phase is called lytic cycle and the non- virulent as temperate or
lysogenic one, and the respective viruses as virulent phage and temperate phage, respectively. The
other temperate lamboid phages are 21, Ø80, Ø81, 424, 434, etc.

15
 Fig 7: Structure of bacteriophage

DNA and Gene Organization of Phage Lambda:

Lambda DNA is a linear and double stranded duplex of about 17 µm in length. It consists
of 48, 514 base pairs of known sequence. Both the ends of 5′ terminus consists of 12 bases which
extend beyond the 3′ terminus nucleotide. This results in single stranded complementary region
commonly called cohesive ends. The cohesive ends form base-pairs and can easily circularize.
Consequently a circular DNA with two single strand breaks are formed. The double stranded
region formed after base pairing of complementary nucleotides is designated as COS. The 12
nucleotides of cohesive ends and process of circularization are shown in Fig. 18.11B-C. The events
of circularization occurs after injection of phage DNA into E.coli cell where the bacterial enzyme,
i.e., E.coli DNA ligase, converts the molecule to a covalently sealed circle.

Life Cycles of Phage Lambda:

Upon adsorption on the lamb receptor of the host cell, lambda gDNA is injected through
the tail which forms a hollow tube through which the DNA passes to the cell. The phage λ leads
two life cycles, the lytic cycle and the lysogenic cycle after injecting its DNA into E.coli cell.
In the lytic cycle, phage genes are expressed and DNA is replicated resulting in production
of several phage particles. The lytic cycle ends with lysis of E.coli cells and liberation of phage
particles. This lytic cycle is a virulent or Sin-temperate where phage multiplies into several particle
(Fig. 18.12).

16
 Fig 7: Multiplication of bacteriophage

In addition, the lysogenic cycle results in integration of phage DNA with bacterial
chromosome and becomes a part of host DNA. It replicates along with bacterial chromosome and
is inherited into progenies. The phage DNA integrated with bacterial chromosome is called
prophage.

The prophage is non-virulent and termed as temperate phage. The bacteria containing
prophage are called lysogenic bacteria, and the prophage stage of viruses as lysogenic viruses.
After treatment of lysogenic bacteria with UV light. X-rays or mitomycin, the prophage can be
separated from bacterial chromosomes and enter the lytic cycle. This process is known as induction
(Fig. 18.12).

17
Genetic Map of Phage Lambda:

The genetic map of phage λ is given in Fig 18.13. The remarkable characteristics of the
map is the clustering of genes according to their functions. For example the head and tail synthesis,
replication and recombination genes are arranged in four distinct clusters. These genes can also be
grouped into three major operons viz. right operon, left operon and immunity operon.
The right operon is involved in the vegetative function of the phage e.g. head synthesis,
tail synthesis and DNA replication leading lytic cycle. The left operon is associated with
integration and recombination events of lysognic cycle.
The immunity operon products interact with DNA and decide whether the phage will
initiate lytic cycle or lysogenic cycle. Singer et al (1977) have given the nucleotide sequence of
ØX174. Genetic map of bacteriophage has been given by Echols and Murialdo (1978).
Function of some important genes is summarized in Table 18.4 and briefly described
below.

Fig 9: Genetic map of bacteriophage

(i) Head Synthesis Genes:

At the left end of phage genome the head genes viz. A, W, B, C, D, E are located which
are associated with phage DNA maturation and head proteins.
(ii) Tail Synthesis Genes:
The genes F, Z, U, V, G, H, M, L, K, I, J are clustered just right to head genes and
code for tail proteins.
(iii) Excision and Integration Genes:

18
 The gene xis codes protein that excises the phage DNA from the bacterial chromosomes,
and int coded protein is involved in integration of phage DNA into the bacterial
chromosome.
(iv) Recombination:
The two genes int and xis codes at att P for site-specific recombination. The three red
genes code for three proteins at normal frequency for general recombination. The redL
codes for exonuclease, red B for beta-protein and red V for gamma protein. The gamma
protein inhibits exonuclease V.
(v) Positive Regulation Gene:
The genes N and R are the positive regulation genes. The proteins code4by these genes
increase the rate of transcription of other genes. Protein coded by N gene induces the
transcription of cll, Q, P, A, red, gam, xis and int, whereas the protein coded by Q gene
stimulates the transcription of head, tail and lysis genes. The N and Q genes are also required
in plaque formation, in the absence of which the number of phage particles would be less but
not zero.

(vi) Negative Regulation Genes:

The cl gene acts as a repressor and its product maintains the prophage in the lysogenic form
in bacterial host. Moreover, the cll and cIII assist the d gene in lysogeny. The proteins
encoded by cro binds to PL and PR and reduce the expression of cl, N, red and xis genes.
The interactions between Q proteins encoded by cro and phage repressor occur in host cell
and the result decides the operation of lytic or lysogenic cycle. The choice between
lysogeny and lysis has been discussed in the preceeding section.

19
(vii) DNA Synthesis Genes:

The two genes O and P are involved in synthesis of phage DNA. The origin of DNA
replication lies within the coding sequence for gene Q which encodes a protein for initiation
of DNA replication, and the gene that generates the cohesive ends is located adjacent to
one of the ends. The function of gene N is required in transcriptional process of these genes.

(viii) Lysis Genes:

The S and R genes control the lysis of bacterial cell envelope which occurs at the end of
lytic cycle.

Choice between Lytic and Lysogenic Cycles:

Soon after circularization of genome and start of transcription, the gpcll and gpcIII
accumulate. The gpcll binds to PRE (promoter for repressor establishment) and stimulates binding
of RNA polymerase (Fig 18.14A). The gpcIII protects gpcll from degradation by host nucleases.
20
 Lambda repressor (gpcl) is rapidly synthesized (B), binds to OL and OR, and inhibits the
synthesis of mRNA and production of gpcll and gpcIII (proteins) (C). The repressor activates the
promoter for repressor maintenance (PRM) which induces c/gene to be transcribed continuously
at a low rate. This process goes on continuously and ensures the stable lysogeny when it is
established (C).
During the course of time the gpcro also accumulates. It binds to OL and OR, turns of the
transcription repressor gene cl and represses PRM function (D). The repressor (gpcl) can block cro
transcription. Therefore, there is a race between the production of gpcl and gpcro proteins.
The detail of this competition is not yet clear but the environmental factors influence the
result of this race for choice of the two cycles. If the repressor wins the competitions, the circular
DNA is inserted into the E. coli genome. The amount of gpcro and the outcome of competition
with gpcl decide the establishment of lysogenic or lytic pathways.

Fig. 10 : Choice between lysis and lysogeny.

2.13. Cloning Vectors: Types & Characteristics

A vector is a DNA molecule which is used for transporting exogenous DNA into the host
cell. A vector is capable of self-replication and stable integration inside the host cell. The
molecular analysis of DNA has been made possible only after the discovery of vectors. The
whole process of molecular cloning involves the following steps:

21
 • Digestion of DNA fragments of the target segment and the vector DNA with the help of
restriction enzymes,
• Ligation of the target segment with the vector DNA with the help of DNA ligases, and
• Introduction of the ligated segment into the host cell for propagation.
General characteristics of a vector:
• It should have an Origin of Replication, known as ori, so that the vector is capable of
autonomous replication inside the host organism.
• It must possess a compatible restriction site for insertion of DNA molecule.
• A vector should always harbour a selectable marker to screen the recombinant organism.
This selectable marker can be an antibiotic resistance gene.
• For easy incorporation into the host machinery, a vector should itself be small in size and
be able to integrate large size of the insert.

2.14. CLONING VECTOR

A cloning vector is also a fragment of DNA which is capable of self-replication and stable
maintenance inside the host organism. It can be extracted from a virus, plasmid or cells of a higher
organism. Most of the cloning vectors are genetically engineered. It is selected based upon the size
and the kind of DNA segment to be cloned.
The cloning vectors must possess the following general characteristics:
• It should small in size.
• It must have an origin of replication.
• It must also be compatible with the host organism.
• It must possess a restriction site.
• The introduction of donor fragment must not intervene with the self-replicating property
of the cloning vector.
• A selectable marker, possibly an antibiotic resistance gene, must be present to screen the
recombinant cells.
• It should be capable of working under the prokaryotic as well as the eukaryotic system.
• Multiple cloning sites should be present.
Importance of Cloning Vectors
Cloning Vectors are used as the vehicle for transporting foreign genetic material into
another cell. This foreign segment of DNA is replicated and expressed using the machinery of the
host organism.
A cloning vector facilitates amplification of a single copy DNA molecule into many copies.
Molecular gene cloning is difficult without the use of the cloning vectors.
History of Cloning Vectors

22
 Herbert Boyer, Keiichi Itakura, and Arthur Riggs were three scientists working in the
Boyer’s lab, University of California, where they recognized a general cloning vector. This cloning
vector had restriction sites for cloning foreign DNA and also, the expression of antibiotic resistance
genes for the screening of recombinant/ transformed cells. The first vector used for cloning
purposes was pBR322, a plasmid. It was small in size, nearly 4kB, and had two selectable markers.
Features of Cloning Vectors
1. Origin of Replication (ori)
• A specific set/ sequence of nucleotides where replication initiates.
• For autonomous replication inside the host cell.
• Foreign DNA attached to ori also begins to replicate.
2. Cloning Site
• Point of entry or analysis for genetic engineering.
• Vector DNA at this site is digested and foreign DNA is inserted with the aid of
restriction enzymes.
• Recent works have discovered plasmids with multiple cloning sites (MCS)
which harbour up to 20 restriction sites.
3. Selectable Marker
• Gene that confers resistance to particular antibiotics or selective agent which,
under normal conditions, is fatal for the host organism.
• Confers the host cell the property to survive and propagate in culture medium
containing the particular antibiotics.
4. Marker or Reporter Gene
• Permits the screening of successful clones or recombinant cells.
• Utilised extensively in blue-white selection.
5. Inability to Transfer via Conjugation
• Vectors must not enable recombinant DNA to escape to the natural population
of bacterial cells.
Essential Characteristics of Cloning Vectors
Regardless of the selection of a vector, all vectors are carrier DNA molecules. These carrier
molecules should have few common features in general such as:

• It must be self-replicating inside host cell.

• It must possess a unique restriction site for RE enzymes.
• Introduction of donor DNA fragment must not interfere with replication
property of the vector.
• It must possess some marker gene such that it can be used for later identification
of recombinant cell (usually an antibiotic resistance gene that is absent in the
host cell).
• They should be easily isolated from host cell.

23
Plasmids
• Plasmids are extra chromosomal circular double stranded DNA replicating elements
present in bacterial cells.
• Plasmids show the size ranging from 5.0 kb to 400 kb.
• Plasmids are inserted into bacterial calls by a process called transformation.
• Plasmids can accommodate an insert size of upto 10 kb DNA fragment.
• Generally plasmid vectors carry a marker gene which is mostly a gene for antibiotic
resistance; thereby making any cell that contains the plasmid will grow in presence of the
selectable corresponding antibiotic supplied in the media.
Phagemids or Phasmid
• They are prepared artificially.
• Phasmid contains the F1 origin of replication from F1 phage.
• They are generally used as a cloning vector in combination with M13 phage.
• It replicates as a plasmid and gets packaged in the form of single-stranded DNA in viral
particles.
Advantages of using Phagemids
• They contain multiple cloning sites.
• An inducible lac gene promoter is present.
• Blue-white colony selection is observed.

Fig 11: Phagemid

24
Cosmids
• Cosmids are plasmids.
• They are capable of incorporating the bacteriophage λ DNA segment. This DNA segment
contains cohesive terminal sites (cos sites).
• Cos sites are necessary for efficient packaging of DNA into λ phage particles.
• Large DNA fragments of size varying from 25 to 45 kb can be cloned.
• They are also packaged into λ This permits the foreign DNA fragment or genes to be
introduced into the host organism by the mechanism of transduction.
Advantages of using cosmids as vectors
• They have high transformation efficiency and are capable of producing a large number of
clones from a small quantity of DNA.
• Also, they can carry up to 45 kb of insert compared to 25 kb carried by plasmids and λ.
Disadvantages of using cosmids as vectors:
• Cosmids cannot accept more than 50 kb of the insert.

Fig 12: Cosmid Vector

Bacteriophage
• The viruses that infect bacteria are called bacteriophage. These are intracellular obligate
parasites that multiply inside bacterial cell by making use of some or all of the host
enzymes.

25
 • Bacteriophages have a very high significant mechanism for delivering its genome into
bacterial cell. Hence it can be used as a cloning vector to deliver larger DNA segments.
• Most of the bacteriophage genome is non-essential and can be replaced with foreign DNA.
• Using bacteriophage as a vector, a DNA fragment of size up to 20 kb can be transformed.

Bacterial artificial chromosomes (BACs)

• Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone
very large DNA fragments ranging in size from 75 to 300 kb.
• BACs basically have marker like sights such as antibiotic resistance genes and a very stable
origin of replication (ori) that promotes the distribution of plasmid after bacterial cell
division and maintaining the plasmid copy number to one or two per cell.
• BACs are basically used in sequencing the genome of organisms in genome projects
(example: BACs were used in human genome project).
• Several hundred thousand base pair DNA fragments can be cloned using BACs.
Advantages of BACs:
• They are capable of accommodating large sequences without any risk of rearrangement.
• BACs are frequently used for studies of genetic or infectious disorders.
• High yield of DNA clones is obtained.
Disadvantages of BACs:
• They are present in low copy number.
• The eukaryotic DNA inserts with repetitive sequences are structurally unstable in BACs
often resulting in deletion or rearrangement.

26
 Fig 13; Bacterial artificial vector

Yeast artificial chromosomes (YACs)

• YACs are yeast expression vectors.
• A very large DNA fragments whose sizes ranging from 100 kb to 3000 kb can be cloned
using YACs.
• Mostly YACs are used for cloning very large DNA fragments and for the physical mapping
of complex genomes.
• YACs have an advantage over BACs in expressing eukaryotic proteins that require post
translational modifications.
• But, YACs are known to produce chimeric effects which make them less stable compared
to BACs.
Advantages of using YACs:
• A large amount of DNA can be cloned.
• Physical maps of large genomes like the human genome can be constructed.
Disadvantages of using YACs:
• Overall transformation efficiency is low.
• The yield of cloned DNA is also low.

27
 Fig 14: Yeast artificial Chromosome
Advantages of BACs over YACs
1. Comparatively stable.
2. Easy to transform.
3. Simple purification required.
4. User- friendly.
5. Aid in the development of vaccines.
Human artificial chromosomes (HACs)
• Human artificial chromosomes (HACs) or mammalian artificial chromosomes (MACs) are
still under development.
• HACs are microchromosomes that can act as a new chromosome in a population of human
cells.
• HACs range in size from 6 to 10 Mb that carry new genes introduced by human researchers.
• HACs can be used as vectors in transfer of new genes, studying their expression and
mammalian chromosomal function can also be elucidated using these microchrosomes in
mammalian system.
Advantages of using HACs:
• No upper limit on DNA that can be cloned.
• It avoids the possibility of insertional mutagenesis.

28
 Fig 15: Human Artificial Chromosome

Other Types of Vectors

All vectors may be used for cloning and are therefore cloning vectors, but there are also
vectors designed specially for cloning, while others may be designed specifically for other
purposes, such as transcription and protein expression.
Expression Vectors
Vectors designed specifically for the expression of the transgene in the target cell are called
expression vectors, and generally have a promoter sequence that drives expression of the
transgene. Expression vectors produce proteins through the transcription of the vector’s insert
followed by translation of the mRNA produced.
Transcription Vectors
Simpler vectors called transcription vectors are only capable of being transcribed but not
translated: they can be replicated in a target cell but not expressed, unlike expression vectors.
Transcription vectors are used to amplify their insert.
Uses of Vectors
Vectors have been developed and adapted for a wide range of uses. Two primary uses are:

(1) To isolate, identify and archive fragments of a larger genome

(2) To selectively express proteins encoded by specific genes.

29
 Vectors were the first DNA tools used in genetic engineering, and continue to be
cornerstones of the technology.
Shuttle vectors:
Shuttle vectors are those which can multiply into two different unrelated species. Shuttle
reactors are designed to replicate in the cells of two
species, as they contain two origins of replication, one
appropriate for each species as well as genes that are
required for replication and not supplied by the host
cell, i.e., it is self-sufficient with the process of its
replication.
The shuttle vectors are of following types:
1. Eukaryotic – Prokaryotic Shuttle Vec-tors:
Vectors that can propagate in eukaryotes and prokaryotes. e.g., YEp vec-tors can be propagated
in yeast (fungi) as well as in E. coli (bacteria).
2. Prokaryotic – Prokaryotic Shuttle Vec-tors:
Vectors that can be propagated in two unrelated prokaryotic host cells, e.g., RSF1010 vectors can
be propagated both in bacteria as well in spirochetes.
The common features of such shuttle vec-tors or eukaryotic vectors are the following:
(a) They are capable of replicating into two or more types of hosts including prokaryotic and
eukaryotic cells.
(b) They replicate autonomously, or integrate into host genome and replicate when the host cell
multiplies.
(c) These vectors are commonly used for transporting genes from one organism to another.

30
 SCHOOL OF BIO AND CHEMICAL ENGINEERING
DEPARTMENT OF BIOTECHNOLOGY

UNIT – 3 – GENETIC ENGINEERING – SBB2102

1
 INTRODUCTION TO R-DNA TECHNOLOGY

1. General strategies for isolation of genomic and plasmid DNA

1.1.DNA Extraction

History of DNA extraction

The first DNA extraction attempt had performed by Friedrich Miescher in 1869. He had
isolated the cell material and named it as the “nuclei” later on his student named it as a “nucleic
acid”. Although he accidentally developed a method for isolation of nucleic acid, he was not sure
that what he isolated was DNA or not. Later on, in 1958 Meselson and Stahl developed a full-
function protocol for DNA extraction. The density gradient centrifugation protocol was the first
protocol described by isolating DNA from E.coli bacteria. The protocol of the proteinase K
enzyme method of DNA extraction was developed by Lahiri and Nurenberger in 1991. They also
modified the protocol by using the Nonidet P40 and SDS. However, the use of proteinase K in
DNA extraction was reported earlier by Miller et al., in 1988.
The phenol-chloroform isoamyl alcohol (PCI) method which is most popular in recent
days was developed by Joseph Sambrook and David W. Russell. The PCI method becomes so
popular that most of the researcher using them in their daily extraction. The yield and consistency
of PCI are much decent.
DNA extraction is required for a variety of molecular biology applications. Figure 1 lists
the basic steps involved in all DNA extraction methods. Many commercial kits are available to
isolate DNA from a variety of biological materials
The basic criteria that any method of DNA isolation from any sample type should meet
include:
(1) efficient extraction of DNA from the sample ,
(2) production of a sufficient amount of DNA for use in downstream processes,
(3) successful removal of contaminants,
(4) isolation of high quality and high purity DNA.
There are three basic steps involved in the process of DNA extraction
1) lysis,
2) precipitation,
3) purification.

2
Step 1 Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside and there
are three ways to do this.
Chemical disruption
Chemicals such as SDS, CTAB, Tris and other detergents can lyse the cell wall/ cell
membrane by solubilizing it. Later DNA dissolves along with cellular proteins.
Enzymatic disruption
Enzymes such as proteinase K, peptidase, protease
disrupt proteins by digesting it. The enzyme works better than
any other chemicals because it directly targets bonds between the
amino acids and digests the protein. Some of the bacteria have a
very smooth and soft cell wall. For example, M.tuberculosis has
a smooth cell wall. By only heating the bacterial solution we can
lyse cell wall.
Mechanical disruption
This can be done with a tissue homogenizer (like
a small blender), with a mortar and pestle, or by cutting the tissue
into small pieces. Mechanical disruption is particularly important
when using plant cells because they have a tough cell wall. Plant
cells have pectin and other polysaccharides present in their cell
wall. This pectin protects the cell from mechanical damage.
Therefore pectin provides additional strength to the cell wall of
the plant.Some of the fungus, algae and bacteria also have hard
cell walls for surviving in harsh conditions. For extracting DNA
from this type of cells, we have to modify protocol with a
combination of mechanical- chemical- enzymatic method.
Once the cell wall or cell membrane lysed, there are no
compartments inside the cell hence all the cell organelles are
mixed into the solution. By doing high-speed centrifugation
DNA remains in the solution and the other cell debris settled into
the bottom of the tube.
Step 2: Precipitation
When you complete the lysis step, the DNA has been freed from the nucleus, but it is now
mixed with mashed up cell parts. Precipitation separates DNA from this cellular debris. First, Na+
ions (sodium) neutralize the negative charges on the DNA molecules, which makes them more
stable and less water soluble. Next, alcohol (such as ethanol or isopropanol) is added and causes
the DNA to precipitate out of the aqueous solution because it is not soluble in alcohol.

3
Step 3: Purification
Now that DNA has been separated from the aqueous phase, it can be rinsed with alcohol
to remove any remaining unwanted material and cellular debris. At this point the purified DNA is
usually re-dissolved in water for easy handling and storage.
Types of DNA extraction methods
Different extraction methods result in different yields and purity of DNA. Some of the
extraction methods have been systematically evaluated for specific applications such as soil and
sediment samples, human microbiome, and fecal samplesDNA extraction methods are broadly
categorized into two categories:
1. Chemical-based DNA extraction method.
2. Solid-phase DNA extraction method.

Fig:1 DNA extraction Methods

Different types of organic and inorganic solutions are used in the chemical or solution-
based DNA extraction method.
The steps of the DNA extraction remain the same in all the types of DNA extraction
methods.SDS, CTAB, phenol, chloroform, isoamyl alcohol, Triton X100, guanidium thiocyanate,
Tris and EDTA are several common chemicals used in the solution based DNA extraction method.

4
Organic Extraction
In this conventional, widely used method, cells are lysed and cell debris is usually removed
by centrifugation. Then, proteins are denatured/digested using a protease, and precipitated with
organic solvents such as phenol, or 1:1 mixture of phenol and chloroform. The protein precipitate
is removed following separation by centrifugation. Purified DNA is usually recovered by
precipitation using ethanol or isopropanol. At some point in the process, RNAs are degraded
through incubation with RNase. In the presence of monovalent cations such as Na+, and at -20°C,
absolute ethanol efficiently precipitates polymeric nucleic acids and leaves behind short-chain and
monomeric nucleic acid components, including the ribonucleotides from RNase treatment in
solution. This method uses hazardous organic solvents, is relatively time-consuming, and residual
phenol or chloroform may affect downstream applications such as PCR. An example of a
commercially available kit that relies on this chemistry is the Easy-DNA® Kit from Thermo
Fisher.
Phenol-chloroform method of DNA extraction:
This method is one of the best methods of DNA extraction. The yield and quality of DNA
obtained by the PCI method is very good if we perform it well. The method is also called as a
phenol-chloroform and isoamyl alcohol, PCI method of DNA extraction.
The major chemicals of PCI DNA extraction methods are lysis buffer, Phenol and
chloroform.The lysis buffer contains Tris, EDTA, MgCl2, NaCl, SDS, and other salts. Here the
components of the lysis buffer help in lysis of cell membrane as well as the nuclear envelope. The
protein portion of the cell denatured with the help of chloroform and phenol which are organic in
nature.
Silica-based technology
Silica-based technologies are widely employed in current kits. DNA adsorbs specifically
to silica membranes/beads/particles in the presence of certain salts and at a defined pH [10]. The
cellular contaminants are removed by wash steps. DNA is eluted in a low salt buffer or elution
buffer. Chaotropic salts are included in the kit buffers to aid in protein denaturation and extraction
of DNA. This method can be incorporated in spin columns and microchips, is cost-effective, has
a simpler and faster procedure than the organic extraction, and is suitable for automation. Kits
based on this method include Purelink Genomic DNA extraction kit from Thermo Fisher and
DNeasy Blood and Tissue Kit from QIAGEN.
Magnetic separation
Magnetic separation is based on DNA reversibly binding to a magnetic solid
surface/bead/particles that have been coated with a DNA binding antibody, or a functional group
that interacts specifically with DNA. After DNA binding, beads are separated from other
contaminating cellular components, washed, and the purified DNA is eluted using ethanol
extraction. This method is rapid, simple to perform and can be automated. However, it can be more
costly than other methodologies. Examples of commercially available kits include the Agencourt
DNAdvance Kit from Beckman Coulter) and Magnetic Beads Genomic DNA Extraction Kit from
Geneaid.

5
Anion exchange technology
DNA extraction by anion exchange chromatography is based on the specific interaction
between negatively charged phosphates of the nucleic acid and positively charged surface
molecules on the substrate. DNA binds specifically to the substrate in the presence of low salt,
contaminants are removed by wash steps using a low or medium salt buffer, and purified DNA is
eluted using a high salt buffer. This technology is most commonly employed in plasmid isolation
kits such as PureLink® HiPure Plasmid DNA Purification Kits from Thermo Fisher, QIAGEN
plasmid mini/midi kits and Genomic-tip, and NucleoBond® PC kits from Macherey Nagel.

Others
Other methods of DNA extraction include salting out, cesium chloride density gradients,
and chelex 100 resin. DNA isolation methods are often modified and optimized for different cell
types or sample sources. For example, cetyltrimethylammonium bromide (CTAB) and guanidium
thiocyanate (GITC) are often included in protocols for DNA extraction from plant materials, and
are discussed in more detail in "DNA extraction from plant tissue and cells".
Purification of DNA
In addition to DNA, a cell extract contains significant quantities of protein and RNA which
can be further purified by following methods
Organic extraction and enzymatic digestion for the removal of contaminants
It involves the addition of a mixture of phenol and chloroform (1:1) to the cell lysate for
protein separation. The proteins aggregate as a white mass in between the aqueous phase
containing DNA and RNA, and the organic layer. Treatment of lysate with pronase or protease, in
addition to phenol/chloroform, ensures complete removal of proteins from the extract. The RNA
can be effectively removed by using Ribonuclease, an enzyme which rapidly degrades RNA into
its ribonucleotide subunits. Repeated phenol extraction is not desirable, as it damages the DNA
Using ion-exchange chromatography
This involves the separation of ions and polar molecules (proteins, small nucleotides and
amino acids) based on their charge. DNA carrying negative charge binds to the cationic resin or
matrix which can be eluted from the column by salt gradient. Gradual increase in salt concentration
detaches molecules from the resin one after another.
Concentration of DNA samples
Concentration of DNA can be done using ethanol along with salts such as sodium acetate,
potassium acetate etc. These salts provide metal ions like sodium ions (Na+), potassium ions (K+)
which help in aggregation and hence precipitation of DNA molecules.

6
Role of chemicals:
Tris: DNA is pH sensitive, Tris buffer maintains the pH of the solution. Also, it interacts with the
lipopolysaccharides of the cell membrane and makes them permeable, this will help in lysis of the
cell membrane.
EDTA: EDTA is a chelating agent and can be used to block DNase activity. DNase is an enzyme
which lyses the DNA. However, every enzyme required cofactor to work properly. The chelator
EDTA blocks the activity of DNase by blocking the cofactor binding site. It will work best in
combination with Tris.
SDS: Sodium dodecyl sulphate is an anionic detergent which helps cell membrane and nuclear
envelope to break open.The SDS removes the negative charges from the amino acid and disrupts
the confirmation of a protein. Therefore, the protein loses its structure and stabilized by using the
SDS.
NaCl: the Na+ ion of NaCl creates the ionic bond with the negative charge of DNA and neutralize
it. It will help DNA comes together and protect from denaturation.
MgCl2: overall, it protects the DNA. MgCl2 blocks the negative charge of the lipoproteins of the
cell membrane. After the lysis of cells, there is no compartment in the cell hence it protects DNA
by mixing with other cell organelles.
Phenol: it precipitates the protein impurities. The combination of phenol, chloroform and isoamyl
alcohol helps in the removal of protein. DNA is insoluble in phenol because phenol is a nonpolar
solution. On the other side, protein has both polar and nonpolar group present in it because of the
long chain of different amino acids. Different amino acids have different groups present on their
side chain. Also, the folding of the protein into the secondary, tertiary and quaternary structure
depends on the polarity of the amino acids. The bonds between amino acids are broken by the
addition of phenol and protein get denatured. Ultimately, we can say the protein becomes unfolded
by addition of phenol. After centrifugation, the phenol settles in the bottom of the tube and DNA
in the aqueous phase while the denatured protein remains between both layers as a whitish cloud.
Lysozyme
● Present in egg-white, salivary secretion and tears.
● Catalyzes the breakdown of cell walls i.e. the peptidoglycan layer.

Chloroform
Chloroform increases the efficiency of phenol for denaturation of the protein. Here,
chloroform allows proper separation of the organic phase and aqueous phase which keeps DNA
protected into the aqueous phase.

7
Isoamyl alcohol
In the phenol-chloroform DNA extraction method, Isoamyl alcohol helps in reducing
foaming between interphase. It prevents the emulsification of a solution. The liquid phase contains
DNA and the organic phase contains lipid, proteins and other impurities. The precipitated protein
denatured and coagulated between both these phases. This will create the cloudy, whitish- foam
between interphase. The anti-foaming agent, isoamyl alcohol stabilized the interphase by removing
the foaming. This will increase the purity of DNA.

1.2.Isolation and Purification of Plasmid DNA

Plasmids are circular, double stranded extra cellular DNA molecules of bacterium and most
commonly used in recombinant DNA technology. The isolation of plasmid DNA
involves three major steps
1. Growth of the bacterial cell.
2. Harvesting and lysis of the bacteria.
3. Purification of the plasmid DNA.
1. Growth of the bacterial cell.
It involves growth of the bacterial cells in a media containing essential nutrients.
2. Harvest and lysis of bacteria
Lysis of bacteria results in the precipitation of DNA and cellular proteins. Addition of
acetate-containing neutralization buffer results in the precipitation of large and less supercoiled
chromosomal DNA and proteins leaving the small bacterial DNA plasmids in solution.
3. Purification of Plasmid DNA
This step is same for both plasmid and genomic but former involves an additional step i.e.
the separation of plasmid DNA from the large bacterial chromosomal DNA.

Methods for separation of plasmid DNA

Separation of plasmid DNA is based on the several features like size and conformation of
plasmid DNA and bacterial DNA. Plasmids are much smaller than the bacterial main
chromosomes, the largest plasmids being only 8% of the size of the E. coli chromosome. The
separation of small molecules (i.e. plasmids) from larger ones (i.e. bacterial chromosome) is based
on the fact that plasmids and the bacterial chromosomes are circular but bacterial chromosomes
break into linear fragments during the preparation of the cell extract resulting in separation of pure
plasmids. The methods of separation of plasmid DNA are described as below,

8
Separation based on size difference

● It involves lysis of cells with lysozyme and EDTA in the presence of sucrose (prevents the
immediate bursting of cells).
● Cells with partially degraded cell walls are formed that retain an intact cytoplasmic
membrane called as spheroplasts.
● Cell lysis is then induced by the addition of a non-ionic detergent (e.g. Triton X100) or
ionic detergents (e.g. SDS) causing chromosomal breakage.
● Bacterial chromosomes attached to cell membranes, upon lysis, get removed with the cell
debris.
● A cleared lysate consisting almost entirely of plasmid DNA is formed with very little
breakage of the bacterial DNA.

Fig 2: Separation of plasmid DNA on the basis of size.

9
Separation based on conformation
Plasmids are supercoiled molecules formed by partial unwinding of double helix of
the plasmid DNA during the plasmid replication process by enzymes called topoisomerases. The
supercoiled conformation can be maintained when both polynucleotide strands are intact, hence
called covalently closed-circular (ccc) DNA. If one of the polynucleotide strands is broken, the
double helix reverts to its normal relaxed state taking an alternative conformation, called open-
circular (oc). Supercoiling is important in plasmid preparation due to the easy separation of
supercoiled molecules from non-supercoiled ones.
The commonly used methods of separation based on conformation are as follows
(a). Alkaline denaturation method
● This method is based on maintaining a very narrow pH range for the denaturation
of non-supercoiled DNA but not the supercoiled plasmid (below figure).
● Addition of sodium hydroxide to cell extract or cleared lysate (pH12.0-12.5)
results in disruption of the hydrogen bonds of non-supercoiled DNA molecules.
● As a result, the double helix unwinds and two polynucleotide chains separate.
● Further addition of acid causes the aggregation of these denatured bacterial DNA
● strands into a tangled mass which can be pelleted by centrifugation, leaving
● plasmid DNA in the supernatant.
Advantage
● Most of the RNA and protein under defined conditions (specifically cell lysis by SDS and
neutralization with sodium acetate) can be removed by the centrifugation.
● No requirement of organic extraction.

10
 Fig 3: Separation of plasmid DNA by Alkaline denaturation method

(b). Ethidium bromide-cesium chloride density gradient centrifugation

● Density gradient centrifugation can separate DNA, RNA and protein. It is a very
efficient method for obtaining pure plasmid DNA.
● A density gradient is produced by centrifuging a solution of cesium chloride at a
very high speed which pulls the CsCl ions towards the bottom. This process is
referred as isopycnic centrifugation.
● The DNA migrates to the point at which it has density similar to that of CsCli.e.1.7
g/cm3 in the gradient.
● In contrast, protein molecules having lower buoyant densities float at the top of the
tube whereas RNA gets pelleted at the bottom.

11
 Density gradient centrifugation in the presence of ethidium bromide (EtBr) can be used to
separate supercoiled DNA from non-super coiled molecules. Ethidium bromide is an intercalating
dye that binds to DNA molecules causing partial unwinding of the double helix. Supercoiled DNA
have very little freedom to unwind due to absence of free ends and bind to a limited amount of
EtBr resulting in very less decrease in buoyant density (0.085 g/cm3 ) than that of linear DNA
(0.125 g/cm3 ). As a result, they form a distinct band separated from the linear bacterial DNA. The
EtBr bound to DNA is then extracted by n-butanol and the CsCl is removed by dialysis.

QUANTIFICATION AND STORAGE OF NUCLEIC ACIDS

Quantification of nucleic acids is done to determine the average concentrations of DNA or
RNA present in a mixture, as well as their purity. The accurate measurement is based on sensitivity,
specificity and interference by contaminants. Various methds that can be employed to quantify the
nucleic acid concentration are listed below,

1. Spectrophotometric analysis
2. Nanodrop
3. Fluorescence based method

Spectrophotometric analysis

Majority of bio-molecules intrinsically absorb light in the ultraviolet and not in the visible
range. This property of UV absorbance can be used to quickly estimate the concentration and
purity of DNA and RNA (also proteins) in a analytical sample. The amount of DNA in a sample
can be estimated by looking at its absorbance at a wavelength of 260nm or 280nm (in the UV
region). Purines and pyrimidines have absorbance maxima slightly below and above 260
respectively. Thus the absorbance maxima of different fragments of DNA vary somewhat
depending on their subunit.composition. Contaminants like proteins exhibit two absorbance peaks,
one between 215-230 nm (due to peptide bonds absorption) and at about 280 nm (absorption by
aromatic amino acids-tyrosine, tryptophan and phenylalanine). Remember that although proteins
have little absorbance at 260 nm, both proteins and nucleic acids absorb light at 280 nm. That is
the reason why, if nucleic acids and proteins are mixed in the same sample, their spectra interfere
(overlap) with one another.
The relationship between concentration of DNA, RNA, Protein and absorptivity are as below:

12
 The purity of a solution of nucleic acid is determined by measuring the absorbance of the
solution at two wavelengths, usually 260 nm and 280 nm, and calculating the ratio of A260/A280.
Value of this ratio is 2.0, 1.8and 0.6 for pure RNA, DNA and protein respectively. A ratio of less
than 1.8 signifies that the sample is contaminated with protein or phenol and the preparation is not
proper.

Nanodrop:
Detection assays are persistently being developed that use progressively smaller amounts
of nucleic acid, often precluding the use of conventional cuvette-based instruments for nucleic acid
quantitation for those that can perform micro-volume quantitation. The patented NanoDrop
microvolume sample retention system functions by combining fiber optic technology and natural
surface tension properties to capture and retain small amounts of sample . This is a novel
technology which allows us to measure nano-liter volumes (pico concentration) of the nucleic acid
(DNA or RNA) sample. It is a type of spectrophotometer with a smaller sample size (as much less
as 1-2 microlitre) requirement and higher sensitivity (even upto pico molar level). This is also a
time saving technology widely used in basic molecular biology research.

13
Fluorometric Quantification:

Fluorometric method applies fluorescence dyes to detect the presence and concentration of
a class of nucleic acid (DNA or RNA). This method is more sensitive and less prone to
contaminants than UV spectroscopy. An assay using Hoechst 33258 dye is specific for DNA
because it is less sensitive to detect RNA. This assay is commonly used for rapid measurement of
low quantities of DNA, with a detection limit of ~1 ng DNA. It is useful for the measurement of
both small and large amounts of DNA (verifying DNA concentrations prior to performing
electrophoretic separations and Southern blots) because this assay accurately quantifies a broad
range of DNA concentrations from10 ng/ml to15 μg/ml. The Hoechst 33258 assay can also be
employed for measuring products of the polymerase chain reaction (PCR) synthesis.
Hoechst 33258 is non-intercalating reagent and binds to the minor groove of the DNA with
a preference for AT sequences (Portugal and Waring, 1988). The binding to the minor groove has
is dependent upon a combination of structural preferences (eg., the minor groove with a series of
contiguous AT base pairs is more narrow).(Neidle (2001) ,Like other minor groove binding
ligands, Hoechst 33258 is positively charged and thus form electrostatic interaction with the
negative potential of stretch of AT base pairs. Upon binding to the minor groove of the double
helix DNA, the fluorescence characteristics of Hoechst 33258 change dramatically, showing a
large increase in emission at ~458 nm.
According to Daxhelet et al, the fluorochrome 4’,6-diamidino-2-phenylindole (DAPI) has
similar characteristics to H33258 and binds to the minor groove as well. DAPI is also appropriate
for DNA or RNA quantitation, although it is not as commonly used as Hoechst 33258. DAPI is
excited with a peak at 344 nm. Emission is detected at around 466 nm for DNA, similar to Hoechst
33258 but for RNA the peak shifts to ~500 nm.

14
Ethidium Bromide Staining:

The IUPAC name for EtBr is 2, 7-diamino-10-ethyl-9-phenylphenanthridiniumbromide. It is

commonly used as a fluorescent dye for nucleic acid staining. It binds as well as intercalates with
nucleic acid (mainly with major and minor groove of DNA) and gives orange fluorescence under
UV radiation from 500 – 590 nm. Usually EtBr may be added in warm agarose gel before
solidification. When DNA or RNA samples are run in agarose gel electrophoresis EtBr molecules
will bind with nucleic acids and help in detection under UV light. The post staining can also be
done for nucleic acid detection.Ethidium bromide (EtBr) is a potent mutagen and carcinogen. Dyes
to stain nucleic acids such as SYBR green, SYBR Safe etc are safer to use instead of EtBr.

Fig4 Ethidium bromide Fig 5 Agarose gel stained with EtBr

Silver Staining:

Silver staining based on reduction of silver nitrate is more sensitive than ethidium bromide for
double stranded DNA, as well as detection of single stranded DNA or RNA with a good sensitivity
(in picogram level). It is based on the reduction of silver cations to insoluble silver metal by nucleic
acids. This chemical reaction is insensitive to the macrostructure of the DNA molecule. Reduced
silver molecules deposit in the gel around the DNA bands, creating a dark black band like image
(i.e. “latent image”). Then the latent image can be developed to visualize by soaking the gel in a
solution of silver cations (Silver nitrate) and a reducing agent (eg. formaldehyde). The silver
granules in the latent image catalyze the further reduction and deposition of silver from the
solution. Bands manifest as dark brown or black regions which appear before significant
background develops. Development is stopped by altering the pH of the gel to a point where silver
reduction is no longer favored.

15
Fig 6: Representation of a silver staining of DNA Lane 1: DNA of lesser concentration Lane 2:
DNA of higher concentration Lane 3: Low molecular weight ladder
Storage of Nucleic Acids

The purified DNA can be stored at -20°C or -70°C under slightly basic conditions (e.g.,
Tris- Cl, pH 8.0) as acidic conditions result in hydrolysis of DNA. Diluted solutions of nucleic
acids can be stored in aliquots and thawed once only. RNA preservation under frozen conditions
is helpful. Purified RNA can be stored at -20°C or -80°C in RNase-free solution such as-

● The RNA Storage Solution (1 mM sodium citrate, pH 6.4 ± 0.2): It is a buffer that delivers
greater RNA stability than 0.1 mM EDTA or TE. The presence of sodium citrate and low
pH minimizes base hydrolysis of RNA. Sodium citrate acts both as a chelating and
buffering agent.
● 0.1 mM EDTA
● TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0)
This RNase-free solution is compatible with all RNA applications including in vitro translation,
reverse transcription, nuclease protection assays and northern analysis.

16
3.2. Strategies for isolation of gene of interest (restriction digestion, PCR)

Gene Cloning: Gene cloning is a process by which large quantities of a specific, desired gene or
section of DNA may be cloned or copied once the desired DNA has been isolated.

Method of Gene Cloning:

1. The gene or DNA that is desired is isolated using restriction enzymes.
2. Both the desired gene and a plasmid are treated with the same restriction enzyme to produce
identical sticky ends.
3. The DNAs from both sources are mixed together and treated with the enzyme DNA ligase to
splice them together.
4. Recombinant DNA, with the plasmid containing the added DNA or gene has been formed.
5. The recombinant plasmids are added to a culture of bacterial cells. Under the right conditions,
some of the bacteria will take in the plasmid from the solution during a process known as
transformation.
6. As the bacterial cells reproduces (by mitosis), the recombinant plasmid is copied. Soon, there
will be millions of bacteria containing the recombinant plasmid with its introduced gene.
7. The introduced gene can begin producing its protein via transcription and translation.

17
Preparation of r-DNA

STEP 1: ISOLATION OF GENE OF INTEREST:

In order to clone a gene the first step is to isolate it using restriction enzymes. These enzymes
recognize specific regions on the DNA molecule. The region of DNA shown below is from
Rhodobacter sphaeroides. The gene of interest lies in the region of the chromosome indicated in
blue. The base sequences are the ones that the restriction enzyme EcoRI recognizes. Note that
reading from left to right in the top strand is the same as reading from right to left in the bottom
strand. Use EcoRI to cut the sugar-phosphate backbone at the points indicated by the red arrows.

18
Unpaired bases result when EcoRI cuts a DNA molecule. Note that the gene of interest is bounded
by fragments of DNA containing unpaired bases or "sticky ends". If the temperature is lowered
and DNA ligase is added these unpaired bases can reanneal following the rules of base pairing.

When pK19 is cut by EcoRI it has "sticky ends" that are complementary to those made by cutting
R. sphaeroides. Like R. sphaeroides the "sticky ends" can reanneal if DNA ligase is added. This
would return the plasmid to it's original ring structure

STEP 2. TRANSFERRING GENE OF INTEREST INTO VECTORS

Cooled, added DNA ligase and the molecules can reanneal. Resulting in a variety of recombinant
forms. One of interest is the plasmid containing the R. sphaeroides DNA.

19
Ligation strategies
In rDNA technology, sealing discontinuities in the sugar-phosphate chains, otherwise
called as ligation, is vital step. This process is catalyzed by DNA ligase by repairing broken
phophodiester bonds. During ligation, the enzyme’s activity is influenced by factors such as 1)
substrate specificity, 2) temperature and 3) salt concentration.

Ligation methods
Joining DNA fragments with cohesive ends by DNA ligase is a relatively efficient process, which
has been extensively used to create artificial recombinants. If the termini of DNA fragments are
not compatible, there are other methods to ligate the fragments.
Cohesive end ligation
The cohesive end ligation is possible when both the foreign DNA to be cloned and the vector DNA
possess the same molecular ends. The compatible sticky ends have been generated by cleavage
with the same enzyme on the same recognition sequences of both foreign DNA and vector DNA.
Using DNA ligase, these molecules can be ligated without any problem. Very often it is necessary
to ligate DNA fragments with different and non-compatible ends, or blunt ends with either
staggered 3' or 5' ends. Incompatible DNA fragments with recessed ends can be ligated by
modifying their ends by any one of the following methods viz., (i) filling in recessed 3' termini
and (ii) renewal of 5' protruding termini.
Blunt end ligation
The E. coli DNA ligase will not catalyze blunt end ligation except under special reaction conditions
of macromolecular crowding. The unique property of T4 DNA ligase was used to ligate DNA
fragments with blunt ends involving short decameric oligonucleotides called linkers.
When same sticky end creating enzyme used for cleavage of vector and gene of interest,
then DNA ligase seals the nick between gene of interest and vector and creates recombinant vector.
Whereas when blunt end creating enzyme used then recoiling become difficult. Moreover, in both
cases of using sticky end and blunt end enzymes, self coiling of vector also occur in high rate
rather than the recombination of vector and genes. These situations are overcome by using

20
i) Linkers

ii) Adaptors

iii) Homopolymer tailing

linkers
Short oligonucleotides (decamers) which contain sites for one or more restriction enzymes are
used to facilitate the ligation process among the DNA fragments with blunt ends.

The linker molecules can be ligated to both ends of the foreign DNA to be cloned and then treated
with restriction endonuclease to produce sticky end fragments which can be incorporated into a
vector molecule that has been cut with the same restriction endonuclease. Insertion by means of
the linker creates restriction sites at each end of the foreign DNA, and thus enables the foreign
DNA excised and recovered after cloning and amplification in the host bacterium.
Adaptors

When linkers added to link at the end of blunt end of gene interest, then there is an
possibility of joining of multiple linkers at the end. This makes some time larger genes and waste

21
of linker molecules. This problem is overcome by using adapters. Since adapters contain only
one end suitable for joining this prevents multiple coiling of adapters. Adapter is a synthetic,
double stranded oligonucleotide used to attach sticky ends to a blunt ended molecule. It contain
normal 5' and 3' end at blunt end and the sticky end of adapter molecule is modified in such manner
that it contain OH group on both 5' and 3' ends. This is achieved by using alkaline phosphatases.
In contrast to linkers, adapters contain preformed sticky ends and joining blunt ends. Because of
lack of 5' phosphate group on sticky end prevents adapter polymer formation. After the adaptors
have been attached the abnormal 5'OH terminus is converted to the natural 5'P form by treatment
with the enzyme polynucleotide kinase, producing sticky ended fragment that can be inserted into
an appropriate vector.

The other strategy adopted for ligating DNA fragments with blunt ends is using adaptors. The
adaptor molecules are synthetic deoxynucleotides that can be used to join two incompatible
cohesive ends, two blunt ends or a combination of both. Such adaptors are of several types viz.,
preformed, conversion and single stranded adaptors.
Preformed adaptors
Preformed adaptors are short DNA duplexes with at least one cohesive end. The problem
of internal cleavage of the insert DNA can be overcome by using a preformed adaptor that will
introduce a new restriction site. For example, an adaptor having BamHI cohesive ends and sites
HpaII and SmaI can be attached to passenger DNA and inserted into a BamHI in vector. After
cloning, passenger DNA can be excised from the hybrid by using any one of the enzymes that
recognize the restriction sites within the adaptor region.

22
Conversion adaptors

Conversion adaptors are synthetic oligonucleotides bearing different cohesive restriction termini.
Such adaptors enable vector molecules that have been cleaved with one endonuclease to be joined
to passenger fragments that have been cleaved with another. Often these adaptors contain internal
restriction sites that permit recovery of the passenger fragment, for example, the EcoRI-BamHI
adaptor contains a site for XhoI.

23
Single stranded

adaptors Single stranded adaptors can be used to make 3’-protruding cohesive ends compatible
with 5’ protruding ends. Such adaptors permit the insertion of passenger fragments into sites on
vectors from which they would otherwise be precluded because of incompatible cohesive ends.

24
Homopolymer tailing

Homopolymer tailing is the other method adopted to clone blunt DNA molecules, especially
cDNA molecules.

The addition of several nucleotides of single type to the 3’ blunt end of DNA molecule is catalyzed
by the enzyme terminal deoxynucleotidyl transferase. The terminal transferase permits the addition
of complementary homopolymer tails (50 to 150 dA or dT long and about 20 dG or dC long) to 3’
end of plasmid vector and passenger DNA. These tails can reanneal to form open circular hybrid
molecules, which can be ligated in vitro or more commonly in vivo following transformations to
produce functional recombinant molecules.

Cloning a Gene (Polymerase Chain Reaction): Clone:

Making exact genetic copies of whole organisms, cells or pieces of DNA are called clones. A clone
is a copy of a plant, animal or micro-organism derived from a single common ancestor cell or
organism. Clones are genetically identical. A gene is said to be cloned when its sequence is
multiplied many times in a common laboratory procedure called polymerase chain reaction (PCR).
PCR copies the cell’s natural ability to replicate its DNA and can generate billions of copies within
a couple of hours.

There are four main stages:

1. The DNA to be copied is heated, which causes the paired strands to separate. The resulting
single strands are now accessible to primers (short lengths of DNA).

25
2. Large amounts of primers were added to the single strands of DNA. The primers bind to
matching sequences along the DNA sequence, in front of the gene that is to be copied. The reaction
mixture is then cooled which allows double-stranded DNA to form again. Because of the large
amounts of primers, the two strands will always bind to primers, instead of to each other.

3. DNA polymerase was added to the mixture. This is an enzyme that makes DNA strands. It can
synthesise strands from all the DNA primer combinations and dramatically increases the amount
of DNA present. One enzyme used in PCR is called Taq polymerase which originally came from
a bacterium that lives in hot springs. It can withstand the high temperature necessary for DNA
strand separation and therefore, can be left in the reaction and still functions.

4. The above steps were repeated until enough DNA is obtained. This whole process is automated
and happens very quickly. The reaction occurs in a small tube which is placed inside a specialised
machine which can make the big temperature adjustments quickly.

Principle of the PCR: The purpose of a PCR (Polymerase Chain Reaction) is to make a huge
number of copies of a gene. This is necessary to have enough starting template for sequencing.

The cycling reactions: There are three major steps in a PCR, which are repeated for 30 or 40
cycles. This is done on an automated cycler7, 8, which can heat and cool the tubes with the reaction
mixture in a very short time. Denaturation at 94C, Annealing at 54C, Extension at 72C.

Fig 7: PCR

26
Because both strands are copied during PCR, there is an exponential increase of the number of
copies of the gene. Let us suppose there is only one copy of the wanted gene before the cycling
starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, and three
cycles will result in 8 copies and so on3

To check whether gene is copied during PCR and to check its right size: Before the PCR
product is used in further applications, it has to be checked if: There is a product formed, the
product is of right size, only one band is formed.

3.3. Selectable Marker Genes and Reporter Genes

The marker genes are of two types:

I. Selectable marker genes.

II. Reporter genes.

Type # I. Selectable Marker Genes:

The selectable marker genes are usually an integral part of plant transformation system. They are
present in the vector along with the target gene. In a majority of cases, the selection is based on
the survival of the transformed cells when grown on a medium containing a toxic substance
(antibiotic, herbicide, antimetabolite). This is due to the fact that the selectable marker gene confers
resistance to toxicity in the transformed cells, while the non- transformed cells get killed.

A large number of selectable marker genes are available and they are grouped into three
categories— antibiotic resistance genes, antimetabolite marker genes, and herbicide resistance
genes (Table 1).

27
 Table 1: List of Selectable Marker Genes

(a) Antibiotic Resistance Genes:

In many plant transformation systems, antibiotic resistance genes (particularly of E. coli) are used
as selectable markers. Despite the plants being eukaryotic in nature, antibiotics can effectively
inhibit the protein biosynthesis in the cellular organelles, particularly in chloroplasts. Some of the
antibiotic resistance selectable marker genes are briefly described.
Neomycin phosphotransferase II (npt II gene):
The most widely used selectable marker is npt II gene encoding the enzyme neomycin
phospho-transferase II (NPT II). This marker gene confers resistance to the antibiotic kanamycin.
The trans-formants and the plants derived from them can be checked by applying kanamycin
solution and the resistant progeny can be selected.

28
Hygromycin phosphotransferase (hpt gene):
The antibiotic hygromycin is more toxic than neomycin and therefore can kill non-transformed
plant cells much faster. Hygromycin phospho-transferase (hpt) gene thus provides resistance to
transformed cells.
Aminoglycoside adenyltransferase (aadA gene):
Aminoglycoside 3′-adenyltransferase (aadA) gene confers resistance to transformed plant cells
against the antibiotics streptomycin and spectionomycin.
(b) Antimetabolite Marker Genes:
Dihydrofolate reductase (dhfr gene):
The enzyme dihydrofolate reductase, produced by dhfr gene is inhibited by the antimetabolite
methotrexate. A mutant dhfr gene in mouse that codes for this enzyme which has a low affinity to
methotrexate has been identified. This dhfr gene fused with CaMV promoter results in a
methotrexate resistant marker which can be used for the selection of transformed plants.
(c) Herbicide Resistance Markers:
Genes that confer resistance to herbicides are in use as markers for the selection of transgenic
plants.
Phosphinothricin acetytransferase (pat/bar gene):
Bialophos, phosphinothricin and glufosinate are commonly used herbicides. The pat/bar genes
code for phosphinothricin acetyltransferase which converts these herbicides into acetylated forms
that are non-herbicidal. Thus, pat/bar genes confer resistance to the transformed plants.
Enolpyruvylshikimate phosphate synthase (epsps/aroA genes):
The herbicide glyphosate inhibits photosynthesis. It blocks the activity of enolpyruvylshikimate
phosphate (EPSP) synthase, a key enzyme involved in the biosynthesis of phenylalanine, tyrosine
and tryptophan. Mutant strains of Agrobacterium and Petunia hybrida that are resistant to
glyphosate have been identified. The genes epsps/aroA confer resistance to transgenic plants which
can be selected.
Bromoxynil nitrilase (bxn gene):
The herbicide bromoxynil inhibits photosynthesis (photosystem II). Bromoxynil nitrilase enzyme
coded by the gene bxn inactivates this herbicide. The gene bxn can be successfully used as a
selectable marker for the selection of transformed plants.

29
Production of Marker-Free Transgenic Plants:
There is a growing concern among the public regarding the use of antibiotic or herbicide resistance
genes as selectable markers of plant transformation:

i. The products of some marker genes may be toxic or allergic.

ii. The antibiotic resistance might be transferred to pathogenic microorganisms in the soil.

iii. There is a possibility of creation of super weeds that are resistant to normally used herbicides.

iv. A transgenic plant with selectable marker genes cannot be transformed again by using the same
selectable markers.

In light of the apprehensions listed above, the public is concerned about the safety of transgenic
technology, particularly related to the selectable marker genes (antibiotic/herbicide resistance
genes). There are fears about the safety of consumption of foodstuffs derived from genetically
engineered plants. This is despite the fact that so far none of the marker genes have been shown to
adversely affect human, animal or environmental safety.
Clean Gene Technology:
The process of developing transgenic plants without the presence of selectable marker genes or by
use of more acceptable marker genes is regarded as clean gene technology. And this will result in
the production of many marker-free transgenic plants that will be readily acceptable by the public.
Some of the approaches for clean gene technology are given.

Avoiding selectable marker genes:

Theoretically, it is possible to totally avoid marker genes and introduce only the transgene of
interest. The transformed paints can then be screened by an advanced technique like polymerase
chain reaction and the desirable plants selected. This approach is not practicable due to cost factor.

Co-transformation with two DNAs:

The transgenic plants can be produced by employing two separate DNAs — one carrying the
desired target gene and the other the marker gene. The transformed plants contain both the genes,
but at different sites on the chromosomal DNA. Traditional breeding techniques (a few rounds)
can be used to get rid of the transgenic plants with selectable markers.

30
Removal of selectable markers:
It is possible to selectively remove the selectable marker genes from the plant genome. For this
purpose, site-specific recombinase systems are utilized. Several recombinase systems are in fact
available which can be used to selectively excise the marker genes from the plant genome.

Cloning of selectable markers between transposable elements:

A selectable marker gene can be cloned between plant transposable elements (Ds elements) and
then inserted. The selectable marker is planked by the sequences that increase the intra-
chromosomal recombination. This results in the excision of the marker gene.

Type # II. Reporter Genes:

A reporter gene may be regarded as the test gene whose expression can be quantified. The plant
transformation can be assessed by the expression of reporter genes (also called as screenable or
scoreable genes). In general, an assay for the reporter gene is carried out by estimating the quantity
of the protein it produces or the final products formed. A selected list of the reporter genes along
with the detection assays is given in Table 49.4, some of the important ones are discussed below.

Table 2: List of Reporter Genes

31
Opine synthase (ocs, nos genes):
The common opines present in T-DNA of Ti or Ri plasmids of Agrobacterium are octopine and
nopaline, respectively produced by the synthase genes ocs and nos. The transformed status of the
plant cells can be easily detected by the presence of these opines. Opines can be separated by
electrophoresis and identified. Alternately, the enzyme activities responsible for the production of
opines can also be assayed.

β-Glucuronidase (gusluidA gene):

β-Glucuronidase producing gene (gusluidA) is the most commonly used reporter gene in assessing
plant transformation for the following reasons:

i. β-Glucuronidase assays are very sensitive.

ii. Quantitative estimation of the enzyme can be done by fluorometric method (using substrate 4-
methylumbelliferryl P-D-glucuronide which is hydrolysed to 4-methylumbelliferone).

iii. Qualitative data on the enzyme can be obtained by histochemical means (enzyme localization
can be detected by chromogenic substance such as substrate X-gluc).

iv. No need to extract and identify DNA.

Green fluorescent protein (gfp gene):

Green fluorescent protein (GFP), coded by gfp gene, is being widely used in recent years. In fact,
in many instances, GFP has replaced GUS since assays of GFP are easier and non-destructive.
Thus, screening of even the primary transplants can be done by GFP which is not possible with
other reporter genes.

Gene for GFP has been isolated from jelly fish Aequorea victoria which is a luminescent organism.
The original gfp gene has been significantly modified to make it more useful as a reporter gene.
GFP emits fluorescence which can be detected under a fluorescent microscope.

Bacterial luciferase (luxA/luxB genes):

The bacterial luciferase genes (luxA and luxB) have originated from Vibrio harveyi. They can be
detected in some plant transformation vectors. The detection assay of the enzyme is based on the

32
principle of bioluminescence. Bacterial luciferase catalyses the oxidation of long-chain fatty
aldehydes that results in the emission of light which can be measured.

Firefly luciferase (luc gene):

The enzyme firefly luciferase, encoded by the gene luc, catalyses the oxidation of D-luciferin (ATP
dependent) which results in the emission of light that can be detected by sensitive luminometers.
The firefly luciferase gene, however, is not widely used as a marker gene since the assay of the
enzyme is rather cumbersome.
Chloramphenicol acetyl transferase (cat gene):
The cat gene producing chloramphenicol acetyl transferase (CAT) is a widely used reporter gene
in mammalian cells. Due to the availability of GUS and GFP reporter systems for plant trans-
formants, CAT is not commonly used. However, some workers continue to use CAT by a sensitive
radioactive assay, for the detection of the reporter gene cat.

33
 SCHOOL OF BIO AND CHEMICAL ENGINEERING
DEPARTMENT OF BIOTECHNOLOGY

UNIT – 4 – GENETIC ENGINEERING – SBB2102

1
 GENE TRANSFER TECHNIQUES

1. Choice of host organism:

A good host should have the following properties:

• Easy to grow and transform.

• Do not hinder replication of recombinant vector.
• Do not have restriction and methylase activities.
• Deficient in recombination function so that the introduced recombinant vector is not
altered.
• Easily retrievable from the transformed host.

Various hosts are used in rDNA technology depending on the goal: For example bacteria, yeast,
plant cells, animal cells, whole plants and animals.

Prokaryotic systems such as E. coli are commonly used due to various advantages like, They
have-

• Well studied expression system,

• Compact genome,
• Versatile,
• Easy to transform,
• Widely available, and
• Rapid growth of recombinant organisms with minimal equipment.

Only disadvantage is that they lack post-translational modification (PTMs) machinery required for
eukaryotic proteins.

Eukaryotic systems are difficult to handle in contrast to bacterial hosts. They are favoured for
expression of recombinant proteins which require post translational modification and only if they
can grow easily in continuous culture.
2. Choice of vector:

Vector is an autonomously replicating (inside a host cell) DNA molecule designed from a
plasmid or phage DNA to carry a foreign DNA inside the host cell. Transformation vectors are of
two types:

2
 • Cloning vector is used increasing the number of copies of a cloned DNA fragment.
• Expression vector is used for expression of foreign gene into a protein.
• If a vector is designed to perform equally in two different hosts, it is called a shuttle vector.

Properties of an ideal vector: A good vector should have the following characteristics:

• Autonomously replicating i.e. should have ori (origin of replication) region.

• Contain at least one selectable marker e. g. gene for antibiotic resistance
• May contain a scorable marker (β-galactosidase, green fluorescent protein etc.)
• Presence of unique restriction enzyme site.
• Have multiple cloning sites.
• Preferably small in size and easy to handle.
• Relaxed control of replication to obtain multiple copies.
• Presence of appropriate regulatory elements for expression of foreign gene.
• High copy number

The selection of a suitable vector system depends mainly on the size limit of insert DNA and the
type of host intended for cloning or expression of foreign DNA

3. Gene Transfer

Introduction

Gene transfer technique is used very widely both in basic research and applied biology.
The delivery of DNA into animal cells is a fundamental and established procedure. It has become
an indispensable tool for gene cloning, the study of gene function and regulation and the production
of small amounts of recombinant proteins for analysis and verification. Gene transfer experiment
helps to express the introduced genetic construct (or transgene) in the recipient cells or to disrupt
or inactivate particular endogenous genes (resulting in a loss of function. There are many
applications of gene transfer like large-scale commercial production of recombinant antibodies
and vaccines and gene medicine or gene therapy. They range from the use of mammalian and
insect cell cultures to transfer of DNA into human patients for the correction or prevention of
disease.

3
 In the organisms or genetically modified whole animals created by Gene transfer every cell
or a specific target population of cells carries a particular alteration. Such animals are used to study
gene function and expression, model human diseases, produce recombinant proteins in their milk
and other fluids, and to improve the quality of livestock herds and other domestic species. The
technology has contributed to understand the functions of the many genes discovered in the
genome projects (functional genomics). Examples of such experiments include systematic DNA-
mediated mutagenesis and gene trap programs in the mouse and in the fruit fly, Drosophila
melanogaster, genome-wide RNA interference experiments in the nematode, Caenorhabditis
elegans, and novel protein interaction screens based on the yeast two hybrid system but performed
using mammalian cells.

Genetic engineering of food is the science which involves deliberate modification of the
genetic material of plants or animals. Introduction of DNA into plants is of great agricultural
potential and medical importance. The gene transfer results can be transient and stable transfection.

Gene therapy can be defined as the deliberate transfer of DNA for therapeutic purposes.
Gene transfer is one of the key factors in gene therapy, and it is one of the key purposes of the
clone. Gene transfer can be targeted to somatic (body) or germ (egg and sperm) cells. In somatic
gene transfer the recipient’s genome is changed, but the change will not be passed on to the next
generation. In germline gene transfer, the parents’ egg and sperm cells are changed with the goal
of passing on the changes to their offspring.

A genetic engineering technique to transfer genes using vectors from one organism to
another or from one cell to another in order to treat disease, construct GMO and economically
important organisms are known as gene transfer. The first evidence of gene transfer was reported
in bacteria by Frederick Griffith in 1928. He had named it a transformation. In 1944, Avery had
demonstrated that the material transfer between bacteria during transformation is the nucleic acid
(DNA) which was later validated by Hershey and Chase in 1952. Although the exogenous gene
transfer in a eukaryotic cell (in vitro) was demonstrated by Rogers in 1970.

The concept of gene transfer between cells was first demonstrated in bacteria, which are capable
of at least four natural forms of genetic exchange. The first mechanism was discovered in 1928 by
Frederick Griffith named as transformation in bacterium Streptococcus pneumoniae. However, he
was unable to determine the nature of the transforming principle. In 1944 Oswald Avery
established that the substance transferred between cells was DNA. A second form of gene transfer

4
was named conjugation discovered by Joshua Lederberg and Edward Tatum in 1946 in Escherichia
coli. This process involved the transfer of DNA through a direct link between the bacterial cells.
In E. coli this conduit between cells took the form of a proteinaceous tube known as a pilus. The
ability of the cells to construct the pilus and pass DNA through it was encoded on a large plasmid
known as the F (for fertility) factor.

In most cases, the act of conjugation involved transfer of the plasmid alone, which became
established in the recipient cell thereby converting it from an F− to an F+ phenotype. In some
cases, however, the F plasmid could integrate into the bacterial chromosome, and conjugation
could result in the transfer of chromosomal genes. This process, which was used to construct the
first genetic map of E. coli, was termed sexduction. Then in 1951, transduction, a new type of gene
transfer mediated by bacteriophage was discovered by Joshua Lederberg and Norton Zinder in
Salmonella. They found that newly formed phage could occasionally package some of the host
cell’s DNA and then transfer it to a second host cell in a subsequent infection.

Two forms of transduction were identified – generalized and specialized transduction. In

generalized transduction the phage head was mistakenly stuffed completely with host cell DNA.
Whereas in specialized transduction the phage genome integrated into the bacterial chromosome
and became linked to host DNA. The fourth mechanism of gene transfer in bacteria is mediated
by complete cell fusion, and occurs in several genera of bacteria including Bacillus and
Streptomyces. Under natural conditions, the viral capsid is needed to introduce the oncogene and
the rest of the genome into an animal host cell. The unusual process of gene transfer without the
viral capsid was named transfection to distinguish it from normal infection.

In bacterial genetics, the term transformation continued to be used to describe the uptake
of naked plasmid or genomic DNA (essentially any DNA which had the potential to transform the
phenotype of the recipient cell) while transfection was used specifically to describe the uptake of
naked phage DNA (or RNA), i.e. nucleic acid which had the potential to initiate a phage replication
cycle. The term transfection became generally accepted to mean the introduction of any sort of
DNA— phage, plasmid, genomic or otherwise—into an animal cell, in the absence of a biological
vector.

5
3.1. Different Gene Transfection Techniques

There are two types of transfection transient and stable. In transient transfection, the
transfected DNA is not integrated into host chromosome. DNA is transferred into a recipient cell
in order to obtain a temporary but high level of expression of the target gene. Stable transfection
is also called permanent transfection. By the stable transfection, the transferred DNA is integrated
(inserted) into chromosomal DNA and the genetics of recipient cells is permanent changed.

Regardless of the delivery method, gene transfer into animal cells must accomplish three
distinct goals. First, the exogenous genetic must be transported across the cell membrane. In
physical transfection methods, transport across the membrane is achieved by direct transfer, where
the membrane is breached during delivery through which DNA and RNA can diffuse. In other
delivery methods, the nucleic acid must form some sort of complex which binds to the cell surface
before internalization. For example, in chemical transfection methods the complex is formed
between nucleic acid and a synthetic compound, while in transduction methods the complex
comprises nucleic acid packaged inside a viral capsid.

Once across the cell membrane, the genetic material must be released in the cell and
transported to its site of expression or activity. Again, the nucleic acid is passive at this stage. In
most transfection methods, DNA or RNA complexes are deposited in the cytoplasm, following
escape from the endosomal vesicle. DNA must be transported to the nucleus, while RNA can
function directly in the cytoplasm. In methods such as particle bombardment and microinjection,
it is possible to deliver DNA directly into the nucleus, so intrinsic transport pathways are not
required. Many viruses also deliver their nucleic acid cargo to the nucleus as part of the infection
cycle, often after interaction with cell surface receptors and either internalization within
endosomes or direct fusion with the plasma membrane. However, few exceptions are there like
poxviruses (e.g. Vaccinia virus) and alphaviruses (e.g. Sindbis virus) which replicate in the
cytoplasm. In the final stage of gene transfer, the exogenous genetic material must be activated. It
must be released from its complex and rendered competent for expression and/or interaction with
the host genome. Exogenous RNA exist only transiently in the host, whereas exogenous DNA can
exist transiently or permanently.

The gene transfer methods normally include three categories: 1. transfection by

biochemical methods; 2. transfection by physical methods; 3. virus-mediately transduction. The
first gene transfer protocols used naked DNA which was mixed with particular chemicals to form

6
synthetic complexes. These synthetic complexes either interact with the cell membrane and
promote uptake by endocytosis, or fuse with the membrane and deliver the DNA directly into the
cytoplasm. Such chemical transfection methods have been widely used. However they are
generally inefficient for gene transfer in vivo. In contrast, physical transfection methods are
efficient for both in vitro and in vivo gene transfer.

Physical transfection methods involve breaching the cell membrane and introducing the
nucleic acid directly into the cell or nucleus. Although there are advantages and disadvantages to
both sets of procedures, some of the most efficient transfection methods in use today involve a
combination of chemical and physical processes. Chemical and physical transfection methods
were first used for the transfer of naked, wild-type viral DNA into animal cells. Now both
techniques are more widely used for the introduction of plasmid vectors and recombinant viral
genomes carrying specific transgenes of interest.

The first transgenic plant was produced via Agrobacterium mediated modified
transformation of Nicotiana tabacum protoplasts by Horsch and co-workers in 1984. Since then
several dozen plant species have been genetically engineered using different techniques.
Simultaneous development of other techniques such as selectable markers facilitated the
development in genetic engineering for obtaining transformed plants. But this technique is not
suitable for monocotyledon plants as they are not natural host of Agrobacterium (there is evidence
that limited gene transfer is possible in monocots by this system). Therefore, other methods of
direct gene transfer have been developed for use with monocots and other species. These can be
categorized.on the basis of the use of protoplasts or cell and tissue as the target materials. The
isolation and purification of protolasts have been presented in earlier Lessons. Freshly isolated
protoplasts are used for genetic transformation using direct gene transfer methods. Let us
understand the procedures of DNA transfer

1. Microinjection

Delivery of nucleic acids to protoplasts or intact cells via microinjection is a labour intensive
procedure that requires special capillary needles, pumps, micromanipulators, inverted microscope
and other equipment. However, injection into the nucleus or cytoplasm is possible and cells can
be cultured individually to produce callus or plants. In this way selection of transformants by drug
resistance or marker genes may be avoided. This method involves skill of the worker to insert
needle into the cytoplasm or in the nucleus. The basic technique is similar to that used for animal

7
cell microinjection. In order to microinject protoplasts or other plant cells, the cells need to be
immobilized (Fig.).

The cells are immobilized by:

1. The use of a holding pipette which holds the cells by vacuum.

2.

Attachment of cells to poly-L-lysine coated cover slips. 3. Embedding the cells in agarose, agar or
sodium alginate.

Glass micropipette are prepared to have openings of about 0.3 uM in diameter and are
inserted into plant cell cytoplasm and nuclei with the aid of a micro manipulators device. A syringe
like device is used for the controlled delivery of volume (10-11 - 10-4 ul) into the plant cell. Most
plant cells are injected while keeping inside microdroplets (2-50 ul) of medium using a chamber
which is sterile, vibration free and permits temperature and humidity regulation. A maximum of
100-200 cells per hour can be microinjected by this method. The recovery of transformants is
dependent upon the regeneration ability of the microinjected cells. Different methods have been
used to grow injured (microinjected) single cells or protoplasts. Hanging droplets, covered under
thin layer of agar or agarose, and micro culture have been used (Fig). Attempts have been made to
inject linear, or super coiled DNA, in cytoplasm or in nucleus. Nuclear injections are found better
for transformations.

Advantages

• No requirement of a marker gene.

• Introduction of the target gene directly into a single cell.

• Easy identification of transformed cells upon injection of dye along with the DNA.

8
 • No requirement of selection of the transformed cells using antibiotic resistance or
herbicide resistance markers.

• It can be used for creating transgenic organisms, particularly mammals.

Disadvantages:

• Only one cell receives DNA per injection.

• Handling of protoplast for microinjection requires skilled persons.
• Sophisticated equipment.
• Requirement of regeneration process from microinjected cells.

2. Particle Gun or Biolistic Method

This is latest technology to transfer DNA into intact tissues. Several devices are developed
using different methods. All to achieve the transfer of micro-sized particles (microprojectiles)
coated with DNA to penetrate the cells. In this procedure micron size tungsten or gold particles
are accelerated in a gun barrel to velocities sufficient for non-lethal penetration of cell walls and
membranes. Klein and co-workers in 1987 developed and used for the first time the particle gun
to transfer chimeric DNA and viral RNA molecules into intact onion cells. Tungsten acted as a
carrier of nucleic acids because it was available in micro-size balls, non-toxic to cells and dense
enough (high density) for rapid penetration of target material. Microprojectile mediated
transformation is a mechanical method of introducing DNA in to any plant species. This method
can be successfully used where plasmids or protoplasts mediated transformation cannot be used.
An acceleration device used to propel particles (micro projectiles) carrying plasmid DNA is called
by various names based on machine or technique used to accelerate the particles such as ‘particle
gun technology’, ‘biolistic method’, ‘DNA bombardment’, ‘particle acceleration of DNA method’
and ‘electric discharge particle acceleration method’.

This is a quick method of stable transformation and testing a gene for cell and organelle specific
expression. This technique has three components –

1. The basic equipment to generate particle acceleration.

2. Metal particles coated with precipitated DNA (desired gene).

3. Plant tissues to be used for particle penetration.

9
The method for regeneration should be previously standardized and proper tissues be selected for
bombardment. (a) Instrument: The instrument is commercially available. Prototype was designed
by Klein and co-workers. It uses the explosive force of gun powder (0.22 caliber gun cartridge) to
accelerate a polypropylene cylindrical macroprojectile. Thin piece of polypropylene
macroprojectile is loaded with microprojectiles coated with DNA. Gun powder explosion forces
this macroprojectile to move with high speed toward another end of barrel, where it is blocked by
a polycarbonate disc having an aperture. Macroprojectile is stopped but microprojectiles move fast
through the aperture towards tissue placed in the same direction. For each transfer, 50 mg tungsten
is accelerated upto 2000 ft per second in a partial vacuum. With this speed, particles reach upto
lower layers of cells in target tissues (Fig). The other devices are similar in basic design concept
but use different methods to accelerate particles like use of compressed air or gas. Compressed air
(130 kg/cm2 pressure) has been used to accelerate microprojectiles at velocities (approximately
440 m/ sec) necessary to achieve DNA delivery to plant cells (Fig). An electric discharge particle
acceleration device differs in basic design from the above described devices. In this device, a high
voltage discharge (14 KV current) delivered to a small water droplet which quickly vaporizes and
releases energy to propel DNA coated gold spheres into target cells (Fig.). In a similar way to
above devices, a DNA carrier is attracted (accelerated) due to potential differences, stopped in-
between by a screen, DNA coated particles cross the screen and fly towards target tissue and
deliver the DNA into cells.

10
 Fig 1: Particle gun or shotgun for delivering DNA coated microprojectiles into plant cells

Fig 2. Compressed air particle acceleration device for delivering DNA coated microprojectiles into
plant cells

Advantages

• Simple and convenient method involving coating DNA or RNA on to gold microcarrier,
loading sample cartridges, pointing the nozzle and firing the device.
• No need to obtain protoplast as the intact cell wall can be penetrated.
• Manipulation of genome of sub-cellular organelles can be done.

11
 • Eliminates the use of potentially harmful viruses or toxic chemical treatment as gene
delivery vehicle.
• This device offers to place DNA or RNA exactly where it is needed into any organism.

Disadvantages

• The transformation efficiency may be lower than Agrobacterium- mediated

transformation.
• Specialized equipment is needed. Moreover the device and consumables are costly.
• Associated cell damage can occur.
• The target tissue should have regeneration capacity.
• Random integration is also a concern.
• Chances of multiple copy insertions could cause gene silencing.

3. Electroporation

Electroporation is a mechanical method used for the introduction of polar molecules

into a host cell through the cell membrane. This method was first demonstrated by Wong
and Neumann in 1982 to study gene transfer in mouse cells. It is now a widely used method
for the introduction of transgene either stably or transiently into bacterial, fungal, plant and
animal cells. It involves use of a large electric pulse that temporarily disturbs the
phospholipid bilayer, allowing the passage of molecules such as DNA.

This method is based on the use of the short electrical pulses of high field strength.
Electroporation causes the uptake of DNA into protoplasts by temporary permeabilization
of the plasma membrane to macromolecules. Protoplasts and foreign DNA are placed in a
buffer between two electrodes and a high intensity electric current is passed (Fig). Electric
field damages membranes and creates pores in membranes. DNA diffuses through these
pores immediately after electric field is applied, until the pore are resealed. The technique
is optimized by using appropriate electric field strength (defined as the applied voltage
divided by the distance between two electrodes).

The optimum field strength is dependent on the following:

1. The pulse length of electric current

12
 2. Composition and temperature of the buffer solution

3. Concentration of foreign DNA in the suspension

4. Protoplasts density, and

5. Size of the protoplasts.

It has been demonstrated that the removal of pectin from the plant wall increases
the amount of DNAwhich can be introduced by electroporation. Tobacco mosaic virus was
introduced in tobacco protoplasts by this method. Electroporation has been used
successfully for transient and stable transformation of protoplasts from a wide range of
species. Plating efficiency (i.e. number of colonies recovered out of number of cells
transferred on plates) of electroporated protoplasts grown on selection medium (containing
selective marker) can be as high as 0.5%. The highest plant transformation efficiencies
have been reported for tobacco, with 0.2% of electroporated leaf mesophyll protoplasts
giving rise to transgenic calli. Low transformation efficiency is common in cereals, e.g. in
rice 0.002% efficiency was recorded.

Fig 3. Top (above) and side view (below) of glass cell with electrodes used for electroporation.

13
Applications

Electroporation is widely used in many areas of molecular biology and in medical field. Some
applications of electroporation include:

• DNA transfection or transformation

Electroporation is mainly used in DNA transfection/transformation which involves introduction of

foreign DNA into the host cell (animal, bacterial or plant cell).

• Direct transfer of plasmids between cells

It involves the incubation of bacterial cells containing a plasmid with another strain lacking
plasmids but containing some other desirable features. The voltage of electroporation creates
pores, allowing the transfer of plasmids from one cell to another. This type of transfer may also be
performed between species. As a result, a large number of plasmids may be grown in rapidly
dividing bacterial colonies and transferred to yeast cells by electroporation.

• Gene transfer to a wide range of tissues

Electroporation can be performed in vivo for more efficient gene transfer in a wide range of tissues
like skin, muscle, lung, kidney, liver, artery, brain, cornea etc. It avoids the vector-specific
immune-responses that are achieved with recombinant viral vectors and thus are promising in
clinical applications.

Advantages

• It is highly versatile and effective for nearly all cell types and species.
• It is highly efficient method as majority of cells take in the target DNA molecule.
• It can be performed at a small scale and only a small amount of DNA is required as
compared to other methods.

Disadvantages
• Cell damage is one of the limitations of this method caused by irregular intensity pulses
resulting in too large pores which fail to close after membrane discharge.

• Another limitation is the non-specific transport which may result in an ion imbalance
causing improper cell function and cell death.

14
3.2. GENE TRANSFER TECHNIQUES: CHEMICAL METHODS

Introduction

Cell membrane is a sheet like assembly of amphipathic molecules that separate cells from their
environment. These physical structures allow only the controlled exchange of materials among the
different parts of a cell and with its immediate surroundings. DNA is an anionic polymer, larger
molecular weight, hydrophilic and sensitive to nuclease degradation in biological matrices. They
cannot easily cross the physical barrier of membrane and enter the cells unless assisted.

Various charged chemical compounds can be used to facilitate DNA transfer directly to the cell.
These synthetic compounds are introduced near the vicinity of recipient cells thereby disturbing
the cell membranes, widening the pore size and allowing the passage of the DNA into the cell.

An ideal chemical used for DNA transfer should have the ability to-

• Protect DNA against nuclease degradation.

• Transport DNA to the target cells.

• Facilitate transport of DNA across the plasma membrane.

• Promote the import of DNA into the nucleus.

The commonly used methods of chemical transfection use the following,

1. Calcium phosphate
2. DEAE dextran
1. Calcium phosphate mediated DNA transfer
Historical perspective

The ability of mammalian cells to take up exogenously supplied DNA from their culture medium
was first reported by Szybalska and Szybalski (1962).
They used total uncloned genomic DNA to transfect human cells deficient for the enzyme
hypoxanthine guanine phosphoribosyl transferase (HPRT). Rare HPRT-positive cells with
fragments of DNA containing the functional gene were identified by selection on HAT medium.
Till then, the actual mechanism of DNA uptake was not understood. It was later found that
successful DNA transfer takes place by the formation of a fine DNA/calcium phosphate co-

15
precipitate, which first settles onto the cells and is then internalized. This technique was first
applied by Graham and Van Der Eb in 1973 for the analysis of the infectivity of adenoviral DNA.

Calcium phosphate transfection

This method is based on the precipitation of plasmid DNA and calcium ions by their
interaction.

Inthis method, the precipitates of calcium phosphate and DNA being small and insoluble can be
easily adsorbed on the surface of cell. This precipitate is engulfed by cells through endocytosis
and the DNA gets integrated into the cell genome resulting in stable or permanent transfection.

Uses

• This method is mainly used in the production of recombinant viral vectors.

• It remains a choice for plasmid DNA transfer in many cell cultures and packaging cell
lines. As the precipitate so formed must coat the cells, this method is suitable only for cells
growing in monolayer and not for suspension cultures.

Fig 4: schematic representation of transfection by Calcium Phosphate Precipitation.

16
Advantages
• Simple and inexpensive
• Applicability to generate stably transfected cell lines
• Highly efficient (cell type dependent) and can be applied to a wide range of cell types.
• Can be used for stable or transient transfection

• Toxic especially to primary cells

• Slight change in pH, buffer salt concentration and temperature can compromise the efficacy
• Relatively poor transfection efficiency compared to other chemical transfection methods
like lipofection.
• Limited by the composition and size of the precipitate.
• Random integration into host cell.
•
Optimal factors (amount of DNA in the precipitate, the length of time for precipitation reaction
and exposure of cells to the precipitate) need to be determined for efficient transfection of the cells.
This technique is simple, expensive and has minimal cytotoxic effect but the low level of transgene
expression provoked development of several other methods of transfection.

2. DEAE-Dextran (Diethylaminoethyl Dextran) mediated DNA transfer

• This method was initially reported by Vaheri and Pagano in 1965 for enhancing the viral
infectivity of cell but later adapted as a method for plasmid DNA transfer.

• Diethylaminoethyl dextran (DEAE-dextran) is a soluble polycationic carbohydrate that

promotes interactions between DNA and endocytotic machinery of the cell.

• In this method, the negatively charged DNA and positively charged DEAE – dextran form
aggregates through electrostatic interaction and form apolyplex. A slight excess of DEAE
– dextran in mixture results in net positive charge in the DEAE – dextran/ DNA complex
formed. These complexes, when added to the cells, bind to the negatively charged plasma
membrane and get internalized through endocytosis. Complexed DNA delivery with
DEAE-dextran can be improved by osmotic shock using DMSO or glycerol.

• Several parameters such as number of cells, polymer concentration, transfected DNA

concentration and duration of transfection should be optimized for a given cell line.
17
Advantages
• Simple and inexpensive
• More sensitive
• Can be applied to a wide range of cell types
• Can be used for transient transfection.

Disadvantages
• Toxic to cells at high concentrations
• Transfection efficiency varies with cell type
• Can only be used for transient transfection but not for stable transfection
• Typically produces less than 10% delivery in primary cells.

Another polycationic chemical, the detergent Polybrene, has been used for the transfection of
Chinese hamster ovary (CHO) cells, which are not amenable to calcium phosphate transfection.
3. Lipofection

• Lipofection is a method of transformation first described in 1965 as a model of cellular

membranes using liposomes.

• Liposomes are artificial phospholipid vesicles used for the delivery of a variety of
molecules into the cells. They may be multi-lamellar or unilamellar vesicles with a size
range of0.1 to 10 micrometer or 20-25 nanometers respectively.

• They can be preloaded with DNA by two common methods- membrane-membrane

fusion and endocytosis thus forming DNA- liposome complex. This complex fuses with
the protoplasts to release the contents into the cell. Animal cells, plant cells, bacteria,
yeast protoplasts are susceptible to lipofection method.

• Liposomes can be classified as either cationic liposome or pH-sensitive.

Cationic liposomes
• Cationic liposomes are positively charged liposomes which associate with the negatively
charged DNA molecules by electrostatic interactions forming a stable complex.
Neutral liposomes are generally used as DNA carriers and helpers of cationic liposomes
due to their non-toxic nature and high stability in serum. A positively charged lipid is often mixed
with a neutral co-lipid, also called helper lipid to enhance the efficiency of gene transfer by
stabilizing the liposome complex (lipoplex). Dioleoylphosphatidyl ethanolamine (DOPE) or
dioleoylphosphatidyl choline (DOPC) are some commonly used neutral co-lipids.

18
 • The negatively charged DNA molecule interacts with the positively charged groups of the
DOPE or DOPC. DOPE is more efficient and useful than DOPC due to the ability of its
inverted hexagonal phase to disrupt the membrane integrity.

• The overall net positive charge allows the close association of the lipoplex with the
negatively charged cell membrane followed by uptake into the cell and then into nucleus.

• The lipid: DNA ratio and overall lipid concentration used in the formation of these
complexes is particularly required for efficient gene transfer which varies with application.
Negatively charged liposomes

• Generally pH-sensitive or negatively-charged liposomes are not efficient for gene transfer.
They do not form a complex with it due to repulsive electrostatic interactions between the
phosphate backbone of DNA and negatively charged groups of the lipids. Some of the
DNA molecules get entrapped within the aqueous interior of these liposomes.

• However, formation of lipoplex, a complex between DNA and anionic lipid scan occur by
using divalent cations (e.g. Ca2+, Mg2+, Mn2+, and Ba2+) which can neutralize the mutual
electrostatic repulsion. These anionic lipoplexes comprise anionic lipids, divalent cations,
and plasmid DNA which are physiologically safe components.
• They are termed as pH sensitive due to destabilization at low pH.
• The efficiency of both in vivo and in vitro gene delivery using cationic liposomes is higher
than that of pH sensitive liposomes. But the cationic liposomes get inactivated and unstable
in the presence of serum and exhibit cytotoxicity. Due to reduced toxicity and interference
from serum proteins, pH-sensitive liposomes are considered as potential gene delivery
vehicles than the cationic liposomes.

19
Liposome Action

Fig 5: Schematic representation of liposome action in gene transfer. (Source: Pleyer U, Dannowski
H. 2002. Delivery of genes via liposomes to corneal endothelial cells. Drug News Perspect, 15(5):
283)

In addition, liposomes can be directed to cells using monoclonal antibodies which

recognize and bind to the specific surface antigens of cells along with the liposomes. Liposomes
can be prevented from destruction by the cell’s lysosomes by pre- treating the cells with chemicals
such as chloroquine, cytochalasin B, colchicine etc. Liposome mediated transfer into the nucleusis
still not completely understood.

Advantages
• Economic

• Efficient delivery of nucleic acids to cells in a culture dish.

• Delivery of the nucleic acids with minimal toxicity.

20
 • Protection of nucleic acids from degradation.

• Measurable changes due to transfected nucleic acids in sequential processes.

• Easy to use, requirement of minimal steps and adaptable to high-throughput systems.

Disadvantages
• It is not applicable to all cell types.

• It fails for the transfection of some cell lines with lipids.

AGRO- BACTERIUM MEDIATED GENE TRANSFER IN PLANTS

Cloning vectors for higher plants were developed in the 1980s and their use has led to the
genetically modified (GM) crops that are in the headlines today. We will examine the genetic
modification of crops and other plants in Chapter 15. Here we look at the cloning vectors and how
they are used. Three types of vector system have been used with varying degrees of success with
Higher plants:
• Vectors based on naturally occurring plasmids of Agrobacterium;
• Direct gene transfer using various types of plasmid DNA;
• Vectors based on plant viruses.

Fig6; Crown gall disease.

Agrobacterium tumefaciens—nature’s smallest genetic engineer

21
Although no naturally occurring plasmids are known in higher plants, one bacterial plasmid, the
Ti plasmid of Agrobacterium tumefaciens, is of great importance. A. tumefaciens is a soil
microorganism that causes crown gall disease in many species of dicotyledonous plants. Crown
gall occurs when a wound on the stem allows A. tumefaciens bacteria to invade the plant. After
infection the bacteria cause a cancerous proliferation of the stem tissue in the region of the crown
(Figure 7.9). The ability to cause crown gall disease is associated with the presence of the Ti (tumor
inducing) plasmid within the bacterial cell. This is a large (greater than 200 kb) plasmid that carries
numerous genes involved in the infective process (Figure 7.10a). A remarkable feature of the Ti
plasmid is that, after infection, part of the molecule is integrated into the plant chromosomal DNA
(Figure 7.10b). This segment, called the T-DNA, is between 15 and 30 kb in size, depending on
the strain. It is maintained in a stable form in the plant cell and is passed on to daughter cells as an
integral part of the chromosomes. But the most remarkable feature of the Ti plasmid is that the T-
DNA contains eight or so genes that are expressed in the plant cell and are responsible for the
cancerous properties of the transformed cells. These genes also direct synthesis of unusual
compounds, called opines, that the bacteria use as nutrients (Figure 7.10c). In short, A. tumefaciens
genetically engineers the plant cell for its own purposes.
Using the Ti plasmid to introduce new genes into a plant cell

It was realized very quickly that the Ti plasmid could be used to transport new genes into plant
cells. All that would be necessary would be to insert the new genes into the T-DNA and then the
bacterium could do the hard work of integrating them into the plant chromosomal DNA. In practice
this has proved a tricky proposition, mainly because the large size of the Ti plasmid makes
manipulation of the molecule very difficult.

The main problem is, of course, that a unique restriction site is an impossibility with a plasmid 200
kb in size. Novel strategies have to be developed for inserting new DNA into the plasmid. Two
are in general use:

• The binary vector strategy (Figure 7.11) is based on the observation that the T-DNA does
not need to be physically attached to the rest of the Ti plasmid. A two-plasmid system, with
the T-DNA on a relatively small molecule, and the rest of the plasmid in normal form, is
just as effective at transforming plant cells. In fact, some strains of A. tumefaciens, and
related agrobacteria, have natural binary plasmid systems. The T-DNA plasmid is small
enough to have a unique restriction site and to be manipulated using standard techniques.

22
• The co-integration strategy (Figure 7.12) uses an entirely new plasmid, based on an E.
coli vector, but carrying a small portion of the T-DNA. The homology between the new
molecule and the Ti plasmid means that if both are present in the same A. tumefaciens cell,
recombination can integrate the E. coli plasmid into the T-DNA region. The gene to be
cloned is therefore inserted into a unique restriction site on the small E. coli plasmid,
introduced into A. tumefaciens cells carrying a Ti plasmid, and the natural recombination

23
process left to integrate the new gene into the T-DNA. Infection of the plant leads to
insertion of the new gene, along with the rest of the T-DNA, into the plant chromosomes.
Production of transformed plants with the Ti plasmid
If A. tumefaciens bacteria that contain an engineered Ti plasmid are introduced into
a plant in the natural way, by infection of a wound in the stem, then only the cells in the

resulting crown gall will possess the cloned gene (Figure 7.13a). This is obviously of little
value to the biotechnologist. Instead a way of introducing the new gene into every cell in
the plant is needed.

24
 There are several solutions, the simplest being to infect not the mature plant but a
culture of plant cells or protoplasts (p. 85) in liquid medium (Figure 7.13b). Plant cells and
protoplasts whose cell walls have re-formed can be treated in the same way as
microorganisms: for example, they can be plated onto a selective medium in order to

isolate transformants. A mature plant regenerated from transformed cells will contain the
cloned gene in every cell and will pass the cloned gene to its offspring. However,
regeneration of a transformed plant can occur only if the Ti vector has been “disarmed” so
that the transformed cells do not display cancerous properties. Disarming is possible
because the cancer genes, all of which lie in the T-DNA, are not needed for the infection
process, infectivity being controlled mainly by the virulence region of the Ti plasmid. In
fact, the only parts of the T-DNA that are involved in infection are two 25 bp repeat
sequences found at the left and right borders of the region integrated into the plant DNA.
25
Any DNA placed between these two repeat sequences will be treated as “T-DNA” and
transferred to the plant. It is therefore possible to remove all the cancer genes from the
normal T-DNA, and replace them with an entirely new set of genes, without disturbing the
infection process.
A number of disarmed Ti cloning vectors are now available, a typical example
being the binary vector pBIN19 (Figure 7.14). The left and right T-DNA borders present
in this vector flank a copy of the lacZ′ gene, containing a number of cloning sites, and a
kanamycin resistance gene that functions after integration of the vector sequences into the
plant chromosome. As with a yeast shuttle vector, the initial manipulations

that result in insertion of the gene to be cloned into pBIN19 are carried out in E. coli, the
correct recombinant pBIN19 molecule then being transferred to A. tumefaciens and thence
into the plant. Transformed plant cells are selected by plating onto agar medium containing
kanamycin.
The Ri plasmid
Over the years there has also been interest in developing plant cloning vectors based
on the Ri plasmid of Agrobacterium rhizogenes. Ri and Ti plasmids are very similar, the
main difference being that transfer of the T-DNA from an Ri plasmid to a plant results not
in a crown gall but in hairy root disease, typified by a massive proliferation of a highly
branched root system. The possibility of growing transformed roots at high density in liquid
culture has been explored by biotechnologists as a potential means of obtaining large
amounts of protein from genes cloned in plants.
Limitations of cloning with Agrobacterium plasmids

26
 Higher plants are divided into two broad categories, the monocots and the dicots.
Several factors have combined to make it much easier to clone genes in dicots such as
tomato, tobacco, potato, peas, and beans, but much more difficult to obtain the same results
with monocots. This has been frustrating because monocots include wheat, barley, rice,
and maize, which are the most important crop plants and hence the most desirable targets
for genetic engineering projects.
The main difficulty stems from the fact that in nature A. tumefaciens and A.
rhizogenes infect only dicotyledonous plants; monocots are outside of the normal host
range. For some time it was thought that this natural barrier was insurmountable and that
monocots were totally resistant to transformation with Ti and Ri vectors, but eventually
artificial techniques for achieving T-DNA transfer were devised. However, this was not
the end of the story. Transformation with an Agrobacterium vector normally involves
regeneration of an intact plant from a transformed protoplast, cell, or callus culture. The
ease with which a plant can be regenerated depends very much on the particular species
involved and, once again, the most difficult plants are the monocots. Attempts to
circumvent this problem have centered on the use of biolistics— bombardment with
microprojectiles (p. 85)—to introduce plasmid DNA directly into plant embryos. Although
this is a fairly violent transformation procedure it does not appear to be too damaging for
the embryos, which still continue their normal development program to produce mature
plants. The approach has been successful with maize and several other important monocots.

27
Fig 8 Cloning genes in plants by direct gene transfer
Biolistics circumvents the need to use Agrobacterium as the means of transferring
DNA into the plant cells. Direct gene transfer takes the process one step further and
dispenses with the Ti plasmid altogether.
Direct gene transfer into the nucleus Direct gene transfer is based on the
observation, first made in 1984, that a supercoiled bacterial plasmid, although unable to
replicate in a plant cell on its own, can become integrated by recombination into one of the
plant chromosomes. The recombination event is poorly understood but is almost certainly
distinct from the processes responsible for T-DNA integration. It is also distinct from the
chromosomal integration of a yeast vector, as there is no requirement for a region of
similarity between the bacterial plasmid and the plant DNA. In fact, integration appears to
occur randomly at any position in any of the plant chromosomes (Figure 7.15). Direct gene
transfer therefore makes use of supercoiled plasmid DNA, possibly a simple bacterial
plasmid, into which an appropriate selectable marker (e.g., a kanamycin resistance gene)
and the gene to be cloned have been inserted. Biolistics is frequently used to introduce the
plasmid DNA into plant embryos, but if the species being engineered can be regenerated
from protoplasts or single cells, then other strategies, possibly more efficient than biolistics,

28
are possible. One method involves resuspending protoplasts in a viscous solution of
polyethylene glycol, a polymeric, negatively charged compound that is thought to
precipitate DNA onto the surfaces of the protoplasts and to induce uptake by endocytosis
(Figure 7.16). Electroporation is also sometimes used to increase transformation frequency.
After treatment, protoplasts are left for a few days in a solution that encourages
regeneration of the cell walls. The cells are then spread onto selective medium to identify
transformants and to provide callus cultures from which intact plants can be grown (exactly
as described for the Agrobacterium system, Figure 7.13b).

Transfer of genes into the chloroplast genome

If biolistics is used to introduce DNA in a plant embryo, then some particles may
penetrate one or more of the chloroplasts present in the cells. Chloroplasts contain their
own genomes, distinct from (and much shorter) than the DNA molecules in the nucleus,
and under some circumstances plasmid DNA can become integrated into this chloroplast
genome. Unlike the integration of DNA into nuclear chromosomes, integration into the
chloroplast genome will not occur randomly. Instead the DNA to be cloned must be flanked
by sequences similar to the region of the chloroplast genome into which the DNA is to be
inserted, so that insertion can take place by homologous recombination (see p. 107). Each
of these flanking sequences must be 500 bp or so in length. A low level of chloroplast
transformation can also be achieved after PEG-induced DNA delivery into protoplasts if
the plasmid that is taken up carries these flanking sequences.
A plant cell contains tens of chloroplasts, and probably only one per cell becomes
transformed, so the inserted DNA must carry a selectable marker such as the kanamycin
resistance gene, and the embryos must be treated with the antibiotic for a considerable
period to ensure that the transformed genomes propagate within the cell. Although this

29
means that chloroplast transformation is a difficult method to carry out successfully, it
could become an important adjunct to the more traditional methods for obtaining GM
crops. As each cell has many chloroplasts, but only one nucleus, a gene inserted into the
chloroplast genome is likely to be expressed at a higher level than one placed in the nucleus.
This is particularly important when the engineered plants are to be used for production of
pharmaceutical proteins (Chapter 13). So far the approach has been most successful with
tobacco but chloroplast transformation has also been achieved with more useful crops such
as soybean and cotton.’

Attempts to use plant viruses as cloning vectors

Modified versions of e and M13 bacteriophages are important cloning vectors for
E. coli (Chapter 6). Most plants are subject to viral infection, so could viruses be used to
clone genes in plants? If they could, then they would be much more convenient to use than
other types of vector, because with many viruses transformation can be achieved simply
by rubbing the virus DNA onto the surface of a leaf. The natural infection process then
spreads the virus throughout the plant. The potential of plant viruses as cloning vectors has
been explored for several years but without great success. One problem is that the vast
majority of plant viruses have genomes not of DNA but of RNA. RNA viruses are not so
useful as potential cloning vectors because manipulations with RNA are more difficult to
carry out. Only two classes of DNA virus are known to infect higher plants, the
caulimoviruses and geminiviruses, and neither is ideally suited for gene cloning.

Caulimovirus
vectors Although one of the first successful plant genetic engineering experiments,
back in 1984, used a caulimovirus vector to clone a new gene into turnip plants, two general
difficulties with these viruses have limited their usefulness. The first is that the total size
of a caulimovirus genome is, like that of e, constrained by the need to package it into its
protein coat. Even after deletion of non-essential sections of the virus genome, the capacity
for carrying inserted DNA is still very limited. Recent research has shown that it might be
possible to circumvent this problem by adopting a helper virus strategy, similar to that used
with phagemids (p. 96). In this strategy, the cloning vector is a cauliflower mosaic virus
(CaMV) genome that lacks several of the essential genes, which means that it can carry a

30
large DNA insert but cannot by itself direct infection. Plants are inoculated with the vector
DNA along with a normal CaMV genome. The normal viral genome provides the genes
needed for the cloning vector to be packaged into virus proteins and spread through the
plant. This approach has considerable potential, but does not solve the second problem,
which is the extremely narrow host range of caulimoviruses. This restricts cloning
experiments to just a few plants, mainly brassicas such as turnips, cabbages, and
cauliflowers. Caulimoviruses have, however, been important in genetic engineering as the
source of highly active promoters that work in all plants and that are used to obtain
expression of genes introduced by Ti plasmid cloning or direct gene transfer.
Geminivirus vectors
What of the geminiviruses? These are particularly interesting because their natural
hosts include plants such as maize and wheat, and they could therefore be potential vectors
for these and other monocots. But geminiviruses have presented their own set of
difficulties, one problem being that during the infection cycle the genomes of some
geminiviruses undergo rearrangements and deletions, which would scramble up any
additional DNA that has been inserted, an obvious disadvantage for a cloning vector.
Research over the years has addressed these problems, and geminiviruses are beginning to
find some specialist applications in plant gene cloning. One of these is in virusinduced gene
silencing (VIGS), a technique used to investigate the functions of individual plant genes.
This method exploits one of the natural defence mechanisms that plants use to protect
themselves against viral attack. This method, called RNA silencing, results in degradation
of viral mRNAs. If one of the viral RNAs is transcribed from a cloned gene contained
within a geminivirus genome, then not only the viral transcripts but also the cellular
mRNAs derived from the plant’s copy of the gene are degraded (Figure 7.17). The plant
gene therefore becomes silenced and the effect of its inactivation on the phenotype of the
plant can be studied.
Viruses as cloning vectors for mammals
For many years it was thought that viruses would prove to be the key to cloning in
mammals. This expectation has only partially been realized. The first cloning experiment
involving mammalian cells was carried out in 1979 with a vector based on simian virus 40
(SV40). This virus is capable of infecting several mammalian species, following a lytic
cycle in some hosts and a lysogenic cycle in others. The genome is 5.2 kb in size (Figure

31
 7.19a) and contains two sets of genes, the “early” genes, expressed early in the infection
cycle and coding for proteins involved in viral DNA replication, and the “late” genes,
coding for viral capsid proteins. SV40 suffers from the same problem as e and the plant
caulimoviruses, in that packaging constraints limit the amount of new DNA that can be
inserted into the genome. Cloning with SV40 therefore involves replacing one or more of
the existing genes with the DNA to be cloned. In the original experiment a segment of the
late gene region was replaced (Figure 7.19b), but early gene replacement is also an option.
In the years since 1979, a number of other types of virus have been used to clone genes in
mammals. These include:

• Adenoviruses, which enable DNA fragments of up to 8 kb to be cloned, longer than is

possible with an SV40 vector, though adenoviruses are more difficult to handle because
their genomes are bigger.
• Papillomaviruses, which also have a relatively high capacity for inserted DNA. Bovine
papillomavirus (BPV), which causes warts on cattle, is particularly attractive because it has
an unusual infection cycle in mouse cells, taking the form of a multicopy plasmid with
about 100 molecules present per cell. It does notcause the death of the mouse cell, and BPV
molecules are passed to daughter cells on cell division, giving rise to a permanently
transformed cell line. Shuttle vectors consisting of BPV and E. coli sequences, and capable
of replication in both mouse and bacterial cells, have been used for the production of
recombinant proteins in mouse cell lines.

32
• Adeno-associated virus (AAV), which is unrelated to adenovirus but often found in the
same infected tissues, because AAV makes use of some of the proteins synthesized by
adenovirus in order to complete its replication cycle. In the absence of this helper virus,
the AAV genome inserts into its host’s DNA. With most integrative viruses this is a random
event, but AAV has the unusual property of always inserting at the same position, within
human chromosome 19. Knowing exactly where the cloned gene will be in the host genome
is important if the outcome of the cloning experiment must be checked rigorously, as is the
case in applications such as gene therapy. AAV vectors are therefore looked on as having
major potential in this area.
• Retroviruses, which are the most commonly-used vectors for gene therapy. Although they
insert at random positions, the resulting integrants are very stable, which means that the
therapeutic effects of the cloned gene will persist for some time

33
34
 SCHOOL OF BIO AND CHEMICAL ENGINEERING
DEPARTMENT OF BIOTECHNOLOGY

UNIT – 5 – GENETIC ENGINEERING – SBB2102

1
 IDENTIFICATION OF GENETIC TRANSFORMANTS AND THEIR APPLICATIONS

1. Selection of recombinants

The need to identify the cells that contain the desired insert at the appropriate and right
orientation and isolate these from those not successfully transformed is of utmost importance to
researchers. Modern cloning vectors include selectable markers (most frequently antibiotic
resistance markers) that allow only cells in which the vector, but not necessarily the insert, has
been transformed to grow. Additionally, the cloning vectors may contain color selection markers
which provide blue/white screening (via alpha-factor complementation) on X-gal medium.
Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in
the cells. Further investigation of the resulting colonies is required to confirm that cloning was
successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA
sequencing.
A host cell having foreign DNA introduced in it is called a transformant. At the end of the
transformation experiment, we get bacterial cells that may contain non-recombinant vector, desired
recombinant vector or undesired recombinant vector or may not contain any vector i.e. non-
transformants. To identify the clone containing desired piece of DNA from among several others,
screening is carried out in two steps:
Selection of clones carrying recombinants
The selection of recombinants is generally done on the basis of marker genes present in the
vector. There are two types of marker genes, selectable marker and a reporter gene or scorable
marker.
Selectable markers
A selectable marker gene codes for a function which enables only those cells which possess
it to survive under suitable conditions. For example, genes conferring resistance to antibiotics like
ampicillin, tetracycline and kanamycin are good selectable markers. When a population of
bacterial cells is plated on an ampicillin containing medium, only those cells that have ampicillin
resistance genes survive and form colonies.
Reporter genes
A reporter gene produces a protein product whose activity can be assayed and permits
either an easy selection or quick identification of cells in which it is present. Therefore it is also
called a scorable marker. Among the more commonly used reporter genes are gus (codes for β
glucuronidase which produces blue colour in the presence of suitable substrate), lux ( luciferase,
produces phosphorescence, gfp (green fluorescence protein, fluoresces on irradiation with U.V.).
Some examples making use of selectable and or scorable markers to identify recombinant clones
are listed below:

2
 i) Insertional inactivation of antibiotic resistance gene This can be explained with the
help of pBR322 which has two selectable markers i.e. Apr and Tcr . Both these genes have unique
cloning sites in them. Insertion of foreign DNA in any of the sites causes inactivation of that gene.
The recombinants thus become susceptible to one of the antibiotics while non-recombinants are
resistant to both.
ii) Insertional inactivation of lacZ gene Some vectors contain a gene or sometimes only
part of a gene, which complements a function missing in their host cells, e.g. lacZ gene ( encodes
fragment of β-galactosidase) in the pUC vectors, M13 and some λ phage vectors which
complements defective lacZ gene (encodes part of β- galactosidase) in E. coli host strains. Insertion
of foreign gene in the vector causes inactivation of lacZ gene. The recombinants are identified by
formation of white colonies /plaques while nonrecombinants form blue colonies /plaques (Fig.11).

Fig 1
iv) Selection on the basis of λ genome size The λ packaging system can only insert DNA
molecules between 38 and 52 kb size into the phage head. Anything less than 38 kb is not
packaged. In some vectors e.g. λ replacement vectors, the size of the vector is less than 38 kb. The
length of DNA insert can be so adjusted as to allow the packaging of only the recombinant DNA.
v) Selection based on Spi (sensitive to phage infection) phenotype Phage λ cannot infect
E.coli cells that have P2 phage integrated in their genome and is said to be Spi+ . Insertion of
foreign DNA causes change in phenotype from Spi+ to Spi− . The recombinants can infect E.coli
having P2 phage while non-recombinants cannot.
3
 vi) Selection based on growth on minimal medium In yeast, an auxotrophic mutant that
has non-functional leu2 gene is used as a host. Such a mutant is able to survive only if leucine is
supplied in the growth medium. However, transformants are able to grow on a minimal medium
(contains no added leucine) due to presence of leu2 gene in the vector (e.g. YEps).
Visual screening
The blue-white screening The blue white screening is one of the most common molecular
techniques that allow detecting the successful ligation of gene of interest in vector (Langley et al.,
1975; Zamenhof & Villarejo 1972; Ausubel et al., 1988). The β-Complementation plasmids are
among the most commonly used vectors for cloning and sequencing the DNA fragments, as they
generally have a good multiple cloning site and an efficient blue-white screening system for
identification of recombinants in presence of a histochemical dye, 5-bromo-4-chloro-3- indolyl-β-
d-galactoside (X-gal), and binding sites for commercially available primers for direct sequencing
of cloned fragments (Manjula 2004). The molecular mechanism for blue/white screening is based
on a genetic engineering of the lac operon in the E. coli as a host cell combined with a subunit
complementation achieved with the cloning vector. The lacZ product, a polypeptide of 1029 amino
acids, gives rise to the functional enzyme after tetramerization (Jacobson et al., 1994) and is easily
detected by chromogenic substrates either in cell lysates or directly on fixed cells in situ (Ko et al.,
1990). The tetramerization is dependent on the presence of the N-terminal region spanning the first
50 residues. Deletions in the N-terminal sequence generate a so-called omega peptide that is unable
to tetramerize and does not display enzymatic activity. The activity of the omega peptide can be
fully restored either in bacteria or in vitro, if a small fragment (called alpha peptide) corresponding
to the intact N-terminal portion is added in trans (Gallagher et al., 1994). The phenomenon is called
α-complementation and the small N-terminal peptide is called alpha peptide. This effect has been
widely exploited for studies in prokaryotes, where special strains that constitutively express omega
peptide exist and allow the detection of expression of the small alpha peptide.
The vector (e.g. pBluescript) encodes the ┙ subunit of LacZ protein with an internal
multiple cloning site (MCS), while the chromosome of the host strain encodes the remaining
omega subunit to form a functional β,-galactosidase enzyme upon complementation. The foreign
DNA can be inserted within the MCS of lacZα gene, thus disrupting the formation of functional
β,-galactosidase. The chemical required for this screen is X-gal, a colorless modified galactose
sugar that is metabolized by β,-galactosidase to form 5-bromo-4-chloroindoxyl which is
spontaneously oxidized to the bright blue insoluble pigment 5,5'-dibromo4,4'-dichloro-indigo and
thus functions as an indicator. Isopropyl β,-D-1- thiogalactopyranoside (IPTG) which functions
as the inducer of the Lac operon, can be used to enhance the phenotype. The hydrolysis of colorless
X-gal by the β,galactosidase causes the characteristic blue colour in the colonies indicating that
the colonies contain vector without insert. White colonies indicate insertion of foreign DNA and
loss of the cells ability to hydrolyze the marker. Bacterial colonies in general, however, are white,
and so a bacterial colony with no vector will also appear white. These are usually suppressed by
the presence of an antibiotic in the growth medium. Blue white screening is thus a quick and easy
technique that allows for the screening of successful cloning reactions through the color of the
bacterial colony. However, the correct type of vector and competent cells are important
considerations when planning a blue white screen. Although the lacZ and many other systems have
been extensively used for gram negative bacteria like E. coli, there are limited options available

4
for screening recombinants transformed in Gram positive bacteria. Chaffin & Rubens (1998) have
developed a gram positive cloning vector pJS3, that utilizes the interruption of an alkaline
phosphatase gene, phoZ, to identify
recombinant plasmids. A multiple cloning site (MCS) was inserted distal to the region
coding for the putative signal peptide of phoZ where the alkaline phosphatase protein expressed
from the phoZ gene (phoZMCS) retained activity similar to that of the native protein and cells
displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-
p). Introduction of any foreign DNA into the MCS of phoZ produced a white colonial phenotype
on agar containing X-p and allowed discrimination between transformants containing recombinant
plasmids versus those maintaining self-annealed or uncut vector. This cloning vector has improved
the efficiency of recombinant DNA experiments in gram-positive bacteria. Cloning inserts into the
multiple cloning region of the pGEM®-Z Vectors disrupts the alpha-peptide coding sequences,
and thus inactivates the beta-galactosidase enzyme resulting in white colonies. Recombinant
plasmids are transformed into the appropriate strain of bacteria (i.e. JM109, DH5), and
subsequently plated on indicator plates containing 0.5 mM IPTG and 40 µg/ml X-gal. A new
version of TA cloning vector with directional enrichment and blue-white color screening has been
reported by Horn (2005).
Limitations of blue-white screening
The "blue screen" technique described above suffers from the disadvantage of using a
screening procedure (discrimination) rather than a procedure for selecting the clones.
Discrimination is based on visually identifying the recombinant within the population of clones on
the basis of a color. The LacZ gene, in the vector used for generating recombinants, may be non-
functional and may not produce β-galactosidase. As a result, these cells cannot convert X-gal to
the blue substance so the white colonies seen on the plate may not be recombinants but just the
background vector.
A few white colonies might not contain the desired recombinant but a small piece of DNA
to be ligated into the vector's MCS might change the reading frame for LacZα, and thus prevent
its expression giving rise to false positive clones. Furthermore, a few linearized vectors may get
transformed into the bacteria, the ends "repaired" and ligated together such that no Lac α is
produced as a result, these cells cannot convert X-gal to the blue substance. On the other hand, in
some cases, blue colonies may contain the insert, when the insert is "in frame" with the LacZα
gene and is devoid of stop codon. This could sometimes lead to the expression of a fusion protein
that is still functional as LacZα. Small inserts which happen to be in frame with the alpha-peptide
coding region may produce light blue colonies, as beta galactosidase activity is only partially
inactivated. Last but not the least, this complex procedure requires the use of the substrate X-gal
which is very expensive, unstable and is cumbersome to use.

5
Reporter gene based screening

Another method for screening and identification of recombinant clones is by using the
green fluorescent protein (GFP) obtained from jellyfish Aequorea victoria. It is a reporter molecule
for monitoring gene expression, protein localization, protein-protein interaction etc. GFP has been
expressed in bacteria, yeast, slime mold, plants, drosophila, zebrafish and in mammalian cells.
Inouye et al., (1997) have described a bacterial cloning vector with mutated Aequorea GFP protein
as an indicator for screening recombinant plasmids. The pGREENscript A when expressed in E.
coli produced colonies showing yellow color in day light and strong green fluorescence under
long-UV. Inserted foreign genes are selected on the basis of loss of the fluorescence caused by
inactivation of the GFP production. The vector used in the study is a derivative of pBluescript
SK(+) (Short et al., 1998) and encodes for the same MCS flanked by T3 and T7 promoters, but
lacks the lacZ gene and the f1 origin region (for single strand DNA production). Instead, the GFP-
S65A cDNA is substituted in its place and is under the control of the lac promoter/operator. The
insertion of foreign DNAs into MCS of pGreenscript A interferes with the production of GFPS65A
and causes a loss in the green fluorescence and yellow color of E. coli colonies. While GFP
solubility appears to be one of the limiting factors in whole cell fluorescence, Davis & Vierstra
(1998) have reported about soluble derivatives of GFP for use in Arabidopsis thaliana.
A system for direct screening of recombinant clones in Lactococcus lactis, based on
secretion of the staphylococcal nuclease (SNase) in the organism, was developed by Loir and co-
workers (Loir et al, 1994). L. lactis strains containing the nuc' plasmids secrete SNase and are
readily detectable by a simple plate test. An MCS was introduced just after the cleavage site
between leader peptide and the mature SNase, without affecting the nuclease activity. Cloning
foreign DNA fragments into any site of the MCS interrupts nuc gene and thus results in nuc mutant
clones which are easily distinguished from nuc' clones on plates. The biggest advantage of this
vector is the possibility of assessing activity of the fusion protein since the nuclease activity is not
diminished by its N-terminal tail and is also reported to be unaffected by denaturing agents such
as sodium dodecyl sulfate (SDS) or trichloroacetic acid.
Limitations of the reporter gene based screening
All the above described plasmids could also result in false positive clones, which is a major
concern for researchers. Loss of GFP fluorescence due to medium composition is also known to
lead to false positive results. Although the SNase based screening would give absolute 100%
recombinants, the active nuc fusion protein expression might render the cell fragile and enhance
its susceptible to the lethal action of the fusion protein upon hyper expression. Also, for all the
above cases, there is a requirement of transfer the genes of interest from the cloning vector to the
expression vectors which calls for fresh cloning followed by screening for recombinants. Hence,
it is evident that the commonly used method for screening and identification of recombinant clones
are associated with problems of false positive results forcing researchers to look for alternate
methods of screening bacterial recombinants and availability of vectors that would act both as
cloning vectors and expression vector are user friendly and advantageous.

6
Indirect methods for clone identification
PCR
Reverse transcription followed by polymerase chain reaction (RT-PCR) can be used for
amplification of RNA sequences in cDNA form. Using gene specific primers, it is possible to clone
specific cDNA molecules from total cellular RNA without need to purify mRNA. Under certain
situations such as when starting DNA material is very small, for example, single cells, it is difficult
to prepare genomic libraries by cell based cloning. In these cases, PCR is the only available
alternative for gene isolation if specific primers are available. However, besides amplifying
specific fragments, PCR can also be used to generate random genomic libraries with the help of
random primers. One limitation of PCR is that the enzyme Taq polymerase can amplify only small
fragments (1-2 kb). In recent years this has been overcome to some extent and it has been possible
to amplify DNA fragments up to 22 kb by method called long PCR.

Screening of transformants for presence of desired gene/gene product

A number of methods have been devised for screening of desired transformants. These
include the following:-
1. Direct selection for the desired gene
2. Identification of the clone from a gene library

Direct selection for the desired gene

In this method, the cloning experiment is designed in such a way that only the desired
recombinant clones are obtained. Selection here occurs at the plating out stage. An example of
direct selection is cloning of genes that specify antibiotic resistance such as kanamycin,
tetracycline or ampicillin resistance. For example, let us consider selection of recombinants that
contain gene for kanamycin resistance. The transformants are plated on agar medium containing
kanamycin. Only the cells that contain the cloned kanamycin resistance gene are able to survive
and form colonies. Hence the technique is called direct selection.
Another situation where direct selection of recombinant clones is done is the marker
rescue technique (Fig. 19). It makes use of auxotrophic mutant strains that have nutritional defects
as the hosts for transformation. For example, suppose an E.coli strain is available which has a
mutation in a gene encoding an enzyme involved in the biosynthesis of amino acid leucine. Such
a mutant strain will only grow in a medium supplemented with leucine. By cloning DNA from a
normal strain i.e. one that can synthesize its own leucine, in the mutant strain and selecting those
transformants that can grow in absence of leucine it is possible to isolate the gene of interest.

7
 Fig 2
Marker rescue is applicable for most genes that encode for biosynthetic enzymes as clones
of these genes can be studied on minimal medium as described for leucine. Auxotrophic strains of
yeast and filamentous fungi are also available and marker rescue has been used to select genes
cloned in these organisms. However, the technique has two limitations: i) a mutant strain must be
available for the gene in question ii) a medium on which only the wild type can survive is needed.

Identification of the clones from a gene library

A library has to be screened in order to find a clone. The identification of a specific

clone from a DNA library can be carried out by exploiting either the sequence of the clone or the
structure/ function of its expressed product. The strategy for screening depends upon information
about the gene of interest, the availability of probe and the cloning method used. One of the key
elements required to identify a gene during screening is a probe. A probe is a piece of DNA or
RNA that contains a portion of the sequence complementary to the desired gene for which we are

8
searching. It is used to detect specific nucleic acid sequences by hybridization (based on
complementarity). The probe can be labeled radioactively (with P32) or nonradioactively (biotin,
digoxigenin and fluorescent dyes etc). Probes can be chemically synthesized based on the amino
acid sequence of the protein coded by the gene. Probes can be homologous or heterologous.
Homologous probe - a probe that is exactly complementary to the nucleic acid sequence
for which we are searching; e. g. a human cDNA used for searching a human genomic library.
Heterologous probe - a probe that is similar to, but not exactly complementary to the
nucleic acid sequence for which we are searching; e.g., a mouse cDNA probe used to search a
human genomic library.
There are several methods for screening DNA libraries. Some of the commonly used
methods are described below:
1. Methods based on nucleic acid hybridization
2. Immunochemical methods
3. Screening DNA libraries using PCR
Methods based on nucleic acid hybridization
● Nucleic acid hybridization is the most commonly used method of library screening first
developed by Grunstein and Hogness in1975 to detect DNA sequences in transformed
colonies using radioactive RNA probes.
● It relies on the fact that a single-stranded DNA molecule, used as a probe can hybridize to
its complementary sequence and identify the specific sequences.
● This method is quick, can handle a very large number of clones and used in the
identification of cDNA clones which are not full-length (and therefore cannot be
expressed).
a) Colony/ plaque hybridization

9
This method was given by Grunstein and Hogness
(1975). It is used for screening both genomic as well
as cDNA libraries and is the most common method
of library screening. The procedure has following
steps:
1. The recombinant bacterial colonies or phage
plaques to be screened are transferred from
the culture plate on to a nitrocellulose filter
paper by replica plating (Fig. 20).
2. The filter with colony replicas is treated with
NaOH to lyse the cells/ phages and to
denature DNA.
3. The filter is then baked at 800 C for two hours
in vacuum oven to fix the DNA.
4. The filter is allowed to hybridize with a
labeled probe.
5. The filter is washed to remove the unbound
excess probe, dried and then subjected to
autoradiography if the probe is radioactively
labeled. The washing conditions are kept
stringent (high temperature and low salt
concentration) when the probe is
homologous and non-stringent (lower
temperature and high salt concentration) in
case of heterologous probe.
Fig 3
Advantages
● This method results in a ‘cleaner’ background and distinct signal (less background probe
hybridization) for λ plaque screening due to less DNA transfer from the bacterial host to
the nitrocellulose membrane while lifting plaques rather than bacterial colonies.
● Multiple screens can be performed from the same plate as plaques can be lifted several
times.
● Screening can be performed at very high density by screening small plaques. High-density
screening has the advantage that a large number of recombinant clones can be screened for
the presence of sequences homologous to the probe in a single experiment.

Probes used for hybridization

Cloned DNA fragments can be used as probes in hybridization reactions if a cDNA clone is
available. DNA or synthetic oligonucleotide probes can be used for identification of a clone from
10
a genomic library instead of RNA probes, for example, to study the regulatory sequences which
are not part of the cDNA clone. A common method of labeling probes is the incorporation of a
radioactive or other marker into the molecule. A number of alternative labeling methods are also
available that involve an amplification process to detect the presence of small quantities of bound
probe and avoid the use of radioactivity. These methods involve the incorporation of chemical
labels such as digoxigenin or biotin into the probe which can be detected with a specific antibody
or the ligand streptavidin, respectively.
Screening by PCR
PCR screening is employed for the identification of rare DNA sequences in complex
mixtures of molecular clones by increasing the abundance of a particular sequence. It is possible
to identify any clone by PCR only if there is available information about its sequence to design
suitable primers.
Preparation of a library for screening by PCR can be done by following ways-

● The library can be plated as plaques or colonies on agar plates and individually inoculated
into the wells of the multi-well plate. However it is a labor intensive process and can lead
to bias in favor of larger colonies or plaques.
● The alternative method involves diluting the library. It involves plating out a small part of
the original library (the packaging mix for a phage library, transformation for a plasmid
library) and calculating the titer of the library. A larger sample is diluted to give a titer of
100 colonies per mL. Dispensing 100 μL into each well theoretically gives 10 clones in
each well. These are then pooled and PCR reactions are carried out with gene-specific
primers flanking a unique sequence in the target to identify the wells containing the clone
of interest. This method is often used for screening commercially available libraries.
Screening methods based on gene expression

Immunological screening
This involves the use of antibodies that specifically recognize antigenic determinants on
the polypeptide. It does not rely upon any particular function of the expressed foreign protein, but
requires an antibody specific to the protein

Earlier immunoscreening methods employed radio-labeled primary antibodies to detect

antibody binding to the nitrocellulose sheet. It is now superseded by antibody sandwiches resulting
in highly amplified signals. The secondary antibody recognizes the constant region of the primary
antibody and is, additionally, conjugated to an easily assayable enzyme (e.g. horseradish
peroxidase or alkaline phosphatase) which can be assayed using colorimetric change or emission
of light using X-ray film.

11
 ● In this technique, the cells are grown as colonies on master plates and transferred
to a solid matrix.
● These colonies are subjected to lysis releasing the proteins which bind to the matrix.
● These proteins are treated with a primary antibody which specifically binds to the
protein (acts as antigen), encoded by the target DNA. The unbound antibodies are
removed by washing.
● A secondary antibody is added which specifically binds to the primary antibody
removing the unbound antibodies by washing.
● The secondary antibody carries an enzyme label (e.g., horse radishperoxidase or
alkaline phosphatase) bound to it which converts colorless substrate to colored
product. The colonies with positive results (i.e. colored spots) are identified and
subcultured from the master plate.

FIGURE 4:Schematic process of immunological screening (a) a nitrocellulose disk is placed onto
the surface of an agar plate containing the phage library. Both agar plate and disk are marked so
as to realign them later. (b) When the nitrocellulose disk is lifted off again, proteins released from
the bacteria by phage lysis bind to the disk. (c) These proteins bind to specific antibody. (d) Plaques
formed by bacteriophage that express the protein bound to the antibody will be detected by
emission of light. The positive clones can be identified by realignment.

12
Figure 5: Schematic process of immunological screening using antibody sandwich.

The main difficulty with antibody-based screening is to raise a specific antibody for each protein
to be detected by injecting a foreign protein or peptide into an animal. This is a lengthy and costly
procedure and can only be carried out successfully with proteins produced in reasonably large
amounts.

Screening by functional complementation

Functional complementation is the process of compensating a missing function in a mutant

cell by a particular DNA sequence for restoring the wild-type phenotype. If the mutant cells are
non-viable, the cells carrying the clone of interest can be positively selected and isolated. It is a
very powerful method of expression cloning and also useful for identification of genes from an
organism having same role as that of defective gene in another organism. The selection and

13
identification of positive clones is based on either the gain of function or a visible change in
phenotype.

For example, the functional complementation in transgenic mice for the isolation of Shaker-2 gene
applied by Probst et al in1988 shown in Figure

Figure 6. Functional complementation in transgenic mice for isolation of Shaker-2 gene

The Shaker-2 mutation is due to the defective gene associated with human deafness disorder.
The BAC clone from the wild type mice are prepared and injected into the eggs of Shaker-2
mutants. The resulting mice are then screened for the presence of wild type phenotype. Thus the
BAC clone carrying the functional Shaker-2 gene is identified which encodes a cytoskeletal
myosin protein. This method can be used for screening human genomic libraries to identify
equivalent human gene.

Drawbacks
● Presence of an assayable mutation within the host cell that can be compensated by the
foreign gene expression which in most cases is not available. In addition, foreign genes
may not fully compensate the mutations.
Applications

14
 ● This method can be used for the isolation of higher-eukaryotic genes (e.g. Drosophila
topoisomerase II gene, a number of human RNA polymerase II transcription factors)
from an organism.
● It can also be possible in transgenic animals and plants to clone a specific gene from its
functional homologue.

Screening DNA libraries using PCR

PCR can be used as alternative to hybridization for screening of genomic and cDNA
libraries. It is possible to identify any clone by PCR but only if there is sufficient information about
its sequence to make suitable primers. To isolate a specific clone, PCR is carried out with gene
specific primers that flank a unique sequence in the target. Pools of clones are maintained in multi-
well plates. Each well is screened by PCR and positive wells are identified. The clones in each
positive well are then diluted into a series in a secondary set of plates and screened again. The
process is repeated until wells having homogenous clones corresponding to the gene of interest
have been identified.

Some important techniques used in characterization of genes/gene products

A number of techniques are used for analyzing the genes/gene products. Some of these
techniques are blotting and hybridization, DNA finger printing and Micro-array analysis. These
are briefly discussed below.

Blotting and hybridization techniques

These techniques are used for detecting a specific gene sequence or its expression, number
of copies of a gene, relatedness among organisms etc. There are three types of blotting techniques:
Southern, Northern and Western blotting.

Southern Blotting
Principle:
● Southern blotting is an example of RFLP (restriction fragment length
polymorphism). It was developed by Edward M. Southern (1975). Southern blotting
is a hybridization technique for identification of particular size of DNA from the
mixture of other similar molecules. This technique is based on the principle of
separation of DNA fragments by gel electrophoresis and identified by labelled probe
hybridization.

15
 ● Basically, the DNA fragments are separated on the basis of size and charge during
electrophoresis. Separated DNA fragments after transferring on nylon membrane,
the desired DNA is detected using specific DNA probe that is complementary to the
desired DNA.
● A hybridization probe is a short (100-500bp), single stranded DNA. The probes are
labeled with a marker so that they can be detected after hybridization.
Procedure/ Steps
Restriction digest: by RE enzyme and amplification by PCR
1. Gel electrophoresis: SDS gel electrophoresis
2. Denaturation: Treating with HCl and NaOH
3. Blotting
4. Baking and Blocking with casein in BSA
5. Hybridization using labelled probes
6. Visualization by autoradiogram

Step I: Restriction digest

● The DNA is fragmentized by using suitable restriction enzyme. RE cuts the DNA
at specific site generating fragments

16
 ● The number of fragments of DNA obtained by restriction digest is amplified by
PCR

Step II: Gel electrophoresis

● The desired DNA fragments is separated by gel electrophoresis
Step III: Denaturation
● The SDS gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to
denature the double stranded DNA fragments.
● DNA strands get separated
Step IV: Blotting
● The separated strands of DNA is then transferred to positively charged membrane
nylon membrane (Nitrocellulose paper) by the process of blotting.
Step V: Baking and blocking
● After the DNA of interest bound on the membrane, it is baked on autoclave to fix
in the membrane.
● The membrane is then treated with casein or Bovine serum albumin (BSA) which
saturates all the binding site of membrane
Step VI: Hybridization with labelled probes
● The DNA bound to membrane is then treated with labelled probe
● The labelled probe contains the complementary sequences to the gene of interest
● The probe bind with complementary DNA on the membrane since all other non-
specific binding site on the membrane has been blocked by BSA or casein.
Step VII: Visualization by Autoradiogram
● The membrane bound DNA labelled with probe can be visualized under
autoradiogram which give pattern of bands.
Application of Southern blotting:
1. Southern blotting technique is used to detect DNA in given sample.
2. DNA finger printing is an example of southern blotting
3. Used for paternity testing, criminal identification, victim identification
4. To isolate and identify desire gene of interest.
5. Used in restriction fragment length polymorphism
6. To identify mutation or gene rearrangement in the sequence of DNA
7. Used in diagnosis of disease caused by genetic defects
8. Used to identify infectious agents

Northern Blotting Technique (Northern Analysis)

17
The term northern blot actually refers specifically to the capillary transfer of RNA from the
electrophoresis gel to the blotting membrane. However, the entire process is commonly referred
to as northern blotting. The northern blot technique was developed in 1977 by James Alwine,
David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its
similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern.
The major difference is that RNA, rather than DNA, is analyzed in the Northern blot.

Northern blotting involves:

● The use of electrophoresis to separate RNA samples according to size and
● Detection of the target sequence using a hybridization probe, whose sequence is
complementary to the part or to the entire target sequence.
Procedure
1. A general blotting procedure starts with extraction of total RNA from a homogenized tissue
sample or from cells.
2.. RNA samples are then separated by gel electrophoresis.
3. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now
separated according to size, are transferred to a nylon membrane through a capillary or vacuum
blotting system.

4. A sheet of nitrocellulose membrane is placed on top of the gel.

5. To ensure good and even contact between gel and membrane, pressure is applied evenly to the
gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane
and gel).

18
6. The transfer-buffer used for the Northern blotting usually contains formamide because it lowers
the annealing temperature of the probe-RNA interaction, thus preventing RNA degradation by
high temperatures.

Buffer-transfer by capillary action from a region of high water potential to a region of low water
potential (usually filter paper and paper tissues) is then used to move the RNA from the gel on to
the membrane; ion exchange interactions bind the RNA. to the membrane due to negative charge
of the RNA and positive charge of the membrane. Once the RNA has been transferred to the
membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. After
a probe has been labeled, it is hybridized to the RNA on the membrane. Experimental conditions
that can affect the efficiency and specificity of hybridization include ionic strength, viscosity,
duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure

19
that the probe has bound specifically and to avoid background signals from arising. The hybrid
signals are then detected by X-ray film and can be quantified by densitometry.

Separation of RNA
The RNA samples are most commonly separated on agarose gels containing formaldehyde as a
denaturing agent for the RNA to limit secondary structure. The gels can be stained with ethidium
bromide (EtBr) and viewed under UV light to observe the quality and quantity of RNA before
blotting. Polyacrylamide gel electrophoresis with urea can also be used in RNA separation but it
is most commonly used for fragmented RNA or micro RNAs.

Detection with Probes

Probes for northern blotting are composed of nucleic acids with a complementary sequence
to all or part of the RNA of interest, they can be DNA, RNA, or oligonucleotides with a minimum
of 25 complementary bases to the target sequence. Commonly, cDNA is created with labelled
primers for the RNA sequence of interest to act as the probe in the northern blot. The probes need
to be labelled either with radioactive isotopes (32P) or with chemiluminescence, which produces
detectable emission of light. X-ray film can detect both the radioactive and chemiluminescent
signals and many researchers prefer the chemiluminescent signals because they are faster, more
sensitive, and reduce the health hazards that go along with radioactive labels. The same membrane
can be probed up to five times without a significant loss of the target RNA.

Applications
1. Northern blotting allows one to observe expression pattern of a particular gene
between tissues, organs, developmental stages, environmental stress levels,
pathogen infection, and over the course of treatment.
2. The expression patterns obtained under given conditions can provide insight into
the function of that gene.
3. Using northern blotting, RNA size can be detected. The quality and quantity of
RNA can be measured on the gel prior to blotting, and the membranes can be stored
and re-probed for years after blotting.

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Western blotting

Principle:
● Western blotting technique is used for identification of particular protein from the
mixture of protein.
● In this method labelled antibody against particular protein is used identify the
desired protein, so it is a specific test. Western blotting is also known as
immunoblotting because it uses antibodies to detect the protein.
Procedure/Steps:
1. Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-
nitro phenyl phosphate which give color.

21
Step I: Extraction of Protein
● Cell lysate is most common sample for western blotting.
● Protein is extracted from cell by mechanical or chemical lysis of cell. This step is
also known as tissue preparation.
● To prevent denaturing of protein protease inhibitor is used.
● The concentration of protein is determined by spectroscopy.
● When sufficient amount of protein sample is obtained, it is diluted in loading buffer
containing glycerol which helps to sink the sample in well.
● Tracking dye (bromothymol blue) is also added in sample to monitor the
movement of proteins.
Step II: Gel electrophoresis
● The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-
acrylamide gel electrophoresis.
● The proteins are separated on the basis of electric charge, isoelectric point,
molecular weight, or combination of these all.
● The small size protein moves faster than large size protein.
● Protein are negatively charged, so they move toward positive (anode) pole as
electric current is applied.
Step III: Blotting
● The nitrocellulose membrane is placed on the gel. The separated protein from gel
get transferred to nitrocellulose paper by capillary action. This type of blotting is
time consuming and may take 1-2 days
● For fast and more efficient transfer of desired protein from the gel to nitrocellulose
paper electro-blotting can be used.
● In electro-blotting nitrocellulose membrane is sandwich between gel and cassette
of filter paper and then electric current is passed through the gel causing transfer of
protein to the membrane.
Step IV: Blocking
● Blocking is very important step in western blotting.
● Antibodies are also protein so they are likely to bind the nitrocellulose paper. So
before adding the primary antibody the membrane is non-specifically saturated or
masked by using casein or Bovine serum albumin (BSA).
Step V: Treatment with Primary Antibody
● The primary antibody (1° Ab) is specific to desired protein so it form Ag-Ab
complex

Step VI: Treatment with secondary antibody

● The secondary antibody is enzyme labelled. For eg. alkaline phosphatase or
Horseradish peroxidase (HRP) is labelled with secondary antibody.

22
 ● Secondary antibody (2° Ab) is antibody against primary antibody (anti-antibody)
so it can bind with Ag-Ab complex.
Step VII: Treatment with suitable substrate
● To visualize the enzyme action, the reaction mixture is incubated with specific
substrate.
● The enzyme convert the substrate to give visible colored product, so band of color
can be visualized in the membrane.
● Western blotting is also a quantitative test to determine the amount of protein in
sample.

Application:
1. To determine the size and amount of protein in given sample.
2. Disease diagnosis: detects antibody against virus or bacteria in serum.
3. Western blotting technique is the confirmatory test for HIV. It detects anti HIV
antibody in patient’s serum.
4. Useful to detect defective proteins. For eg Prions disease.
5. Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis B and
Herpes\

Dot Blot and Slot Blot Hybridization

These two techniques represents the simplification of Southern and Western blots saving
the time involved in procedures of chromatography, electrophoresis, restriction digestion and
blotting of DNA or proteins from the gel to membrane. Here nucleic acid mixture is directly
applied (blotted) on to the nylon or nitrocellulose membrane where hybridization between probe
and target takes place, denatured to single-stranded form and baked at 80°C to bind DNA target to
membrane. In dot-blot, target is blotted as circular blots whereas in slot-blots, it is in the form of
rectangular blots. Due to this, slot-blot offers greater precision in observing different hybridization
signals. After blotting, membrane is allowed to dry and non-specific sites are blocked by soaking

23
in blocking buffer containing BSA. It is then followed by hybridization of labeled probe for
detection of specific sequences or gene.

Figure:Dots and slots in dot and slot blot hybridization

These procedures can only detect presence and absence of particular sequence or gene. It cannot
distinguish between two molecules of different sizes as they appear as single dot on membrane. It
also has application in detecting alleles that differ in single nucleotide with the help of allele-
specific oligonucleotides.

Important Examples of Transgenic Animal

The following points highlight the three important examples of transgenic animal. The examples
are: 1. Cloning Dolly 2. Transgenic Mice 3. Reporter System.

1. Cloning Dolly:

In February 1996, Ian Wilmut and co-workers from the Roslin Institute and PPL Therapeutics,
both in Edinburgh, Scotland, reported in Nature journal that they had successfully cloned a sheep
from a cell taken from the udder of a six-year old ewe.

The cloned lamb was named Dolly. In the past, genetically identical animal embi70s had been
created only with amphibian cells, and those created from adult nuclei had never successfully
reached adulthood. Cloning in which the nuclei came from fetal cells or cells from cell lines had
been successful before in mammals. The word clone means to create a genetically identical copy.

24
To clone an animal, it is necessary to begin with an egg, the only cell known to initiate and support
development. In order to clone an individual, it is necessary to get an egg without a nucleus and
then to transplant in it a nucleus of known origin.

Techniques for nuclear transplantation had been worked out with frogs and toads in the 1950s. The
Scottish scientists succeeded in obtaining sheep eggs, enucleating them (removing their nuclei)
and then transferring in donor nuclei by fusing the donor cells and the enucleated eggs with an
electric pulse.

The electric pulse also initiated development of the egg. Although only one pregnancy of the
twenty nine initiated was successful, the lamb that was born seemed normal in every respect; since
it had produced offspring.

Others had tried this type of experiments with many types of animals, including mice. They were
not successful for numerous reasons. The most likely explanation for the recent success, according
to scientists, is that the donor cells were kept in a non-growth phase for several days, which may
have synchronized them with the oocyte.

Thus the nucleus and the oocyte were at the same stage of cell cycle and thus compatible. Other
reorganization that had to take place in the donor chromosomes are not really known for certain
but one thing is clear: the nucleus of an adult cell in the sheep has all of the genetic material needed
to support normal growth and development of the egg. The work has since been repeated with
goats, cattle and mice.

There are various ramifications to the success of this work:

1. Mammal cloning could becomes a routine procedure. This would allow us to study mammalian
development and to replicate genetically identical individuals, particularly transgenic animals that
would have particular genome of value.

2. We can also use these techniques to study aging, since an “old” nucleus in initiating development
of a new organism.

25
3. Also, of interest is the interaction of a particular genome with a particular cytoplasm, since the
cytoplasm contains not only the material needed for early development, but also cell organelles,
including mitochondria that have their own genetic material.

Transgenic Mice:

● Animal cells, like the protoplasts of plant cells, can take up foreign chromosomes or DNA
directly from the environment with a very low efficiency (in the presence of calcium
phosphate). Directly injecting the DNA greatly improves the efficiency.
● For example, transgenic mice are now routinely prepared by injecting DNA either into
oocytes or one or two- celled embryos obtained from female mice after appropriate
hormonal treatment.
● After injection of about 2 picoliters (2 X 10-12 liters) of cloned DNA, the cells are
reimplanted into the uteruses of receptive female hosts. In about 15% of these injections,
the foreign DNA incorporates into the embryo.
● Transgenic animals are used to study the expression and control of foreign eukaryotic
genes. In 1988, a transgenic mouse prone to cancer was first genetically engineered animal
to be patented. Thus mouse provides an excellent model for studying cancer. (A
controversy arose as to whether engineered higher organisms should be patentable;
currently they are).
● Mice have already been successfully transfected with a rat growth-hormone gene, and
transgenic sheep have been produced that express the gene for a human clotting factor. The
latest recombinant DNA dispute arose, from cloning of sheep in 1997.
● The transfection can also be mediated by retroviruses (RNA viruses containing the gene
for reverse transcriptase). For example, a retroviral vector was ritrodused and repaired
human white blood cell lacking the enzyme adenosine deaminase.
● A retrovirus responsible for a form of leukemia in rodents, the Moloney Murine leukemia
viruses was engineered so that all the virus genes were removed and replaced with an
antibiotic marker (neomycin resistance) and the human adenosine deaminase gene.
● The virus binds to the cell surface and is taken into the cell, its RNA is converted to DNA
by reverse transcription and the DNA is incorporated into one of the cells, chromosomes.
It is not possible for the highly modified virus to attack and damage the cells.

26
Reporter System:

● Two reporter systems are used to indicate that a transfection experiment was successful.
Plants can be transfected with the Ti plasmids of Agrobacterium tumefaciens. When a plant
is infected with A. tumefaciens containing the Ti plasmid, a crown gall tumor is induced
transferring the T- DNA region.
● Those cells transfected with the T-DNA are induced to grow as well as to produce opines
that the bacteria feed on. Much recent research has concentrated on engineering Ti
plasmids to contain other genes that are also transferred to the host plants during infection,
creating transgenic plants. One series of experiments have been especially charming.
● Tobacco plants have been transinfected by Ti plasmids containing the luciferase gene from
fireflies. The product of this gene catalyzes the ATP- dependent oxidation of luciferin,
which emits light. When a transfected plant is watered with luciferin, it glows like a firefly.
The value of these experiments is not the production of glowing plants but rather the use
of the glow to “report” the action of specific genes.
● In further experiments, the promoters and enhancers of certain genes were attached to the
luciferase gene. As a result, luciferase would only be produced when these promoters were
activated; thus, the glowing areas of the plant show where the transfected gene is active.
● One of the more recent reported systems developed uses a gene from jellyfish that produces
green fluorescent protein. The value of this system is that it “reports” when ultra-violet
light falls on it, rather than it requiring an addition, as in the luciferase system (see Tamarin,
2002).

Examples of Transgenic Plants

The following points highlight the top six examples of transgenic plants. The examples are: 1.
“High Lysine” Corn 2. Enhanced Nitrogen Fixation 3. Herbicide-Tolerant Plants 4. Disease-
Insect-Resistant Varieties 5. Male Sterility 6. Transgenic Plants as Bioreactors (Molecular
Farming).

1. “High Lysine” Corn:

○ The proteins stored in plant seeds function as reserves of amino acids used during
seed germination and pre- emergence growth of the young seedling. Plant seed

27
 storage proteins also provide the major source of proteins in the diets of most
humans and herbivorous higher animals.
○ Worldwide, the seeds of legumes and cereal grains are estimated to provide humans
with 70 per cent of their dietary requirements. Unfortunately, the major seed storage
proteins of cereals, called prolamines (Zeins in com or maize), are virtually lacking
in the amino acid lysine.
● Since prolamines account for about half of the total protein content of cereal seeds, diets
based largely on cereal grains will be deficient in lysine (an essential amino acid). In the
case of com, the seed proteins are also deficient in tryptophan (another essential amino
acid) and to a lesser extend, methionine (an essential amino acid).
● Because of the importance of cereal seeds as human and animal foods, plant breeders have
attempted for several decades to develop varieties with increased lysine, tryptophan and
methionine content.
● In com, mutants such as opaque-2, sugary-1 and floury-2 have increased amounts of lysine
and/or methionine in seeds, but these mutant strains have undesirable soft kernels and
produce lower yields. These “high lysine” mutant strains all result from mutations that alter
the relative proportions of different seed storage proteins.
● In general, they lower the prolamine (zein) content so that other seed proteins account for
a larger proportion of the total seeds proteins. This, in turn, increases the relative amounts
of lysine and/or methionine in the seeds.
● Several genes of corn encoding zeins have now been cloned and sequenced. With this
information in hand, researchers have suggested that it might be possible to produce “high
lysine” corn by genetic engineering. Since the zeins have no known enzymatic functions,
one might be able to modify zein genes by mutagenesis without inflicting any deleterious
effects on function(s).
● Specifically site-specific mutagenesis could be used to introduce more lysine codons into
zein sequences. Then, these “high lysine” zein coding sequences could be joined to strong
promoters such as the CaMV35S promoter and reintroduced into com plants by
transformation by means of electroporation or a microjectile gun.
● However, a possible difficulty in engineering “high lysine” com by this method is that the
modified zein proteins might not package properly in seed storage structures.

28
 ● The zein proteins are synthesized on rough endoplasmic reticulum and they aggregate
within this membranous structure into dense deposits called protein bodies. The formation
of protein bodies is thought to involve hydrophobic and weak polar interactions between
the zein monomers.
● If so, charged amino acids such as lysine might interfere with proper packaging of zeins
during protein body formation. In 1988, B.A. Larkin and colleagues have introduced new
lysine and tryptophan codon into a zein cDNA by oligonucleotide-directed site-specific
mutagenesis.
● When BNA transcripts of these modified cDNAs were translated efficiently and the “high
lysine” zein products were found to self-aggregate into dense structures similar to those
present during polar body formation in com. These results offer encouragements that “high
lysine” com might indeed be produced by means of genetic engineering.

2. Enhanced Nitrogen Fixation:

Plants are only able to utilize nitrogen that has been incorporated into chemical compounds such
as ammonia, urea, or nitrates. No green plant is capable of extracting diatomic nitrogen (N2)
molecules directly from the atmosphere. Although plants use only a small fraction of the total
nitrogen pool, they are dependent on a continuous supply of nitrogen in usable form (most often
called “fixed nitrogen”).

● On-going fixation of atmospheric nitrogen is required because the fixed nitrogen in soil is
constantly being depleted by leaching, by utilization for the growth of plants and
microorganisms and by denitrifying bacteria that converts fixed nitrogen back to N2. As a
result, millions of dollars/rupees is spent each year on nitrogen fertilizers in order to obtain
optimal yields of major crops such as com and the cereal grains.
● Biological nitrogen fixation is the alternative to the use of the industrially fixed nitrogen
provided in fertilizers. Several species of bacteria and lower algae are capable of converting
N2 to fixed forms of nitrogen that can be utilized by plants.
● Because the purchase of nitrogen fertilizers represent one of the major expenses incurred
with current agricultural production methods, a major effort has been made and continues
to be devoted to the development of enhanced methods of biological nitrogen fixation.

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 ● Certain free-living soil bacteria such as Azotobacter vinelandii and Klebsiella pneumoniae
directly convert atmospheric nitrogen to ammonia. These bacteria are an important source
of fixed nitrogen and in addition, have proven to be extremely valuable subjects for studies
on the mechanism of nitrogen fixation.
● In Klebsiella, there are 17 nif (nitrogen fixation) genes organised in seven operons. The
complexity of the nitrogen fixation metabolic machinery in these bacteria has important
implications for anyone who might aspire to engineer nitrogen-fixing plants.
● The situation with nitrogen fixation is very different from that of herbicide tolerance. It is
one thing to construct a single chimeric gene and transfer that gene to plants, but it is far
more difficult to engineer 17 different chimeric genes, to transfer all of them to the same
recipient plant, and to coordinate their expression in the plant so that all the components of
the complex nitrogen-fixing enzymatic machinery are synthesized in proper amounts and
in the appropriate cells of the plant.
● At present, the possibility of engineering nitrogen-fixing plants is largely fantasy, but
remember that travelling to the moon was pure science fiction not too many years ago.

Some facts about nitrogen fixation:

The phenomenon of fixation of atmospheric nitrogen by biological means is known as diazotrophy

or biological nitrogen fixation and these prokaryotes as diazotrophs or nitrogen fixers. For the first
time, Beijerinck (1888) isolated Rhizobium from root nodules of leguminous plants. Thereafter, S
.Winogradsky discovered a free-living nitrogen fixing bacterium, Clostridium pasteurianum.

● Then a large number of nitrogen fixers were discovered from different sources and
associations. For example, Frankia from nodules of non-legumes (e.g., alder, Casuarina,
etc.), Nostoc from lichens, Anabaena from Azolla leaves, and coralloid roots of Cycas. The
diazotrophs may be free living or in symbiotic form.
● Heterocysts are the sites of nitrogen fixation in some cyanobacteria, e.g., Anabaena,
Nostoc, etc. Heterocysts are formed in the absence of utilizable combined nitrogen, such
as ammonia because it inhibits heterocyst differentiation and N2-fixing enzyme, the
nitrogenase. Heterocysts lack oxygen evolving photosystem II, ribulose biphosphate and
may lock photosynthetic biliproteins.

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 ● Chlorophyll-a is present in heterocysts. Wall of heterocyst contains O2 binding glycolipids
which together with respiratory consumption maintain the anaerobic conditions (i.e.,
highly reduced atmosphere) necessary for N2 fixation. In contrast, vegetative cells adjacent
to heterocysts, both photosystem I and II are present; therefore, oxygen evolution takes
place by these cells.
● Other cyanobacteria, that lack heterocyst also do N2-fixation, e.g., Oscillatoria (see Dubey
2006).
● Another very important source of biologically fixed nitrogen is the symbiotic relationship
between bacteria of the genus Rhizobium and plants of family Leguminosae (the alfalfa,
clovers, soyabeans, peanuts, peas, etc.).
● This symbiotic nitrogen fixation occurs in highly differentiated root nodules that develop
when Rhizobium bacteria interacts with the roots of legumes. Nodule formation is
dependent on genetic information of both the plants and the bacterium. The nitrogenase
that catalyzes N2 reduction is encoded by the bacterial genome, but the fixed nitrogen is
utilised for growth of both bacteria and the host plants.
● Once the mechanisms responsible for establishing this symbiotic relationship and for
nodule formation are known, and the genes that control these processes have been
identified, it might be possible to use genetic engineering to modify non-legume plants
(e.g., com, rice and wheat, such that they will participate in similar symbiotic relationships
with nitrogen-fixing bacteria.
● However, once again, this will undoubtedly be a challenging task because the genetic
control of nodule formation is clearly complex. Nevertheless, experiments are in progress
with goals of modifying bacteria so as to enhance their nitrogen-fixing capacity and to
broaden their host range to include additional plant species.

3. Herbicide-Tolerant Plants:

The development of herbicide-tolerant varieties of agronomically important plants such as com,

soybeans and the cereas promises to have a major impact on agriculture, both economically and
on production practices. Weeds compete with crops for soil nutrients and routinely lead to
significant losses in yield.

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 ● Modern agriculture makes use of herbcides to control weeds and minimize the losses.
Unfortunately, the available herbicides seldom provide the degree of specifications that is
desired, and most herbicides will control only certain classes and not others.
● Broad-spectrum herbicides may give good weed control, but in doing, so usually have
deleterious effects on the growth of crop plants as well. As a result, scientists are now
considering alternate approaches to weed control.
● The most promising of the alternate approaches is the development of herbicide-tolerant
plant varieties for use with broad-spectrum or totally nonspecific herbicides. Obviously,
the potential economic value of herbicide-tolerant plant varieties is significant.
● Herbicides are simple chemical compounds that kill or inhibit the growth of plants without
deleterious effects on animals. Herbicides usually inhibit the processes that are unique to
plants, for example, photosynthesis.
● Most frequently, herbicides act as inhibitors of essential enzyme reactions. Thus, anything
that diminishes the level of inhibition will provide increased herbicide tolerance.

The two most common sources of herbicide tolerance are:

(1) Over-production of the target enzyme and

(2) Mutations resulting in enzymes that are less sensitive to the inhibitor (usually due to a lower
affinity of the enzyme for the inhibitor).

● It seems likely that the most successful strategy for developing herbicide- tolerant plants
will be to combine both sources of tolerance, that is, to engineer plants that overproduce
herbicide-tolerant mutant enzymes. We can consider, here, the example of glyphosate.
● Glyphosate is one of the most potent broad-spectrum herbicide known; it is marketed under
the trade name Roundup Glyphosate acts by inhibiting the enzyme 5-enolpyruvylshikimate
3-phosphate synthase (EPSP synthase), an essential compound in the biosynthesis of the
aromatic amino acids tyrosine, phenylalanine and tryptophan.
● These aromatic amino acids are essential components in the diets of higher animals since
the enzymes that catalyse the biosynthesis of these amino acids are not present in higher
animals. Therefore, since higher animals contain EPSP synthase, glyphosate has no toxic
effects on animal systems. In this respect, glyphosate is an ideal herbicide.

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 ● Glyphosate of herbicide, does inhibit the EPSP synthases of microorganisms as well as
those of plants. By selecting for growth in the presence of glyphosate concentrations that
inhibit the growth of wild-type bacteria, researchers have been able to isolate glyphosate-
tolerant mutants of Salmonella typhimurium, Aerobacter aerogenes and Escherichia coli.
● In bacteria, EPSP synthase is encoded by the aroA gene. When the mutant bacterial aroA
genes were provided with plant promoters and polyadenylation signals (producing,
chimeric genes) and were introduced into plants, the transgenic plants exhibited increased
tolerance to glyphosate (herbicide).
● In plants, synthesis of aromatic amino acids takes place in chloroplasts, but the genes
encoding the biosynthetic enzymes such as EPSP synthase are nuclear genes. The
translation products contain a transit peptide that targets the protein to the chloroplasts.
● This transit peptide is then cleaved off proteolytically upon entering the chloroplasts to
yield the active enzyme. Experiments have now shown that the petunia transit peptide will
target the E. coli aroA gene product into tobacco chloroplasts and will produce glyphosate
tolerance in the recipient cell lines.

4. Disease-Insect-Resistant Varieties:

● Several microorganisms and certain native plants produce proteins that are toxic to specific
plant pathogens, both microbial pathogens and insects that feed on plants. One goal of plant
genetic engineering is to transfer the genes encoding these protein toxins to agronomically
important plants with the hope that expressing the toxin genes in these plants will provide
biological control Disease-insect of at least some plant diseases and insect pests.
● Currently, plant diseases resistant plants and insect pests are controlled almost exclusively
by the use of broad- spectrum chemical bacteriocides, fungicides and insecticides.
However, there is reason for concern about the potential damage to ecosystems and
pollution of groundwater that might result from the widespread use of these chemicals on
agricultural crops. Thus, scientists are searching for alternate methods for controlling these
pathogens.
● The best-known example of the use of natural gene products to control plant pests are the
insect toxins of Bacillus thuringiensis. Each of the toxin genes of B. thuringiensis encodes
a large protein that aggregate, to form protein crystals in spores and these protein crystals
are highly toxic to certain insects.

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 ● Some of the insects that are killed by these protein toxins are plant pests of major economic
importance. Different subspecies of B. thuringiensis produce toxins that kill different
insects. For example, the toxin produced by B. thuringiensis subspecies kurstaki kills
lepidopteran larvae such as the tobacco hornworm.
● The gene that encodes this toxin has been isolated and shown to synthesize a functional
toxin in E. coli. A chimeric gene with the structure CaMV35S promoter/B. thuringiensis
subspecies kurstaki toxin coding sequence/Ti nos 3′ termination sequence was constructed.
● This chimeric gene was placed in a Ti vector, and tomato leaf disc cells were transformed
by co-cultivation with A .tumefaciens harboring the engineered Ti vector-chimeric gene
construct. Transgenic tomato plants were regenerated and shown to express the chimeric
gene. The toxicity of the gene-product synthesized in the transgenic plants was tested by
allowing tobacco hornworm larvae to feed on the transgenic plants and on control plants.
● All the larvae applied to the transgenic plants died within a few days; larvae applied to the
control plants remained healthy and eventually consumed the entire plants. These results
support the feasibility of using genetic engineering to produce transgenic tomato. Pathogen
resistant plant varieties.

BT Cotton:

● The transgenic technology provides alternative and innovative method to improve pest
control management which are ecofriendly, effective, sustainable and beneficial in terms
of yield. The first genes available for genetic engineering of crop plants for pest resistance
were cry genes (popularly known as Bt genes) from bacterium Bacillus thuringiensis.
These genes are specific to particular group of insect pests and are not harmful to other
useful insects such as butterfly, silk worms and honeybee.
● Transgenic crops (e.g., cotton, rice, maize, potato, tomato, brinjal, cauliflowers, cabbage,
etc.) with Bt genes have been developed and such transgenetic variety proved effective in
controlling insect pests and it has been claimed worldwide that it has led to significant
increase in yield along with dramatic reduction in pesticide use.
● The most notable example is Bt cotton (which contain cry/Ac gene) that is resistant to a
notorious insect pest bollworm (Helicoperpa armigera). Bt cotton was adopted for mass
cultivation in India in year 2002.

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5. Male Sterility:

● Male sterile plants are very important to prevent unnecessary pollination and to eliminate
the process of emasculation during the production of hybrid plants. Such sterile male plants
are created by introducing a gene coding for an enzyme (barnase), which is an RNA
hydrolyzing enzyme) that inhibits pollen formation. This gene is expressed specifically in
the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only
to the cells involved in pollen production.

6. Transgenic Plants as Bioreactors (Molecular Farming):

● Plants are amazing and cheap chemical factories that need only water, minerals, sunlight
and carbon dioxide to produce thousand types of chemical molecules (see Dubey 2006).
Given the right genes, plants can serve as bioreactors to new compounds such as amino
acids, proteins, vitamins, plastics, pharmaceuticals (peptides and proteins), drugs, and
enzymes for food industries and so on.

Thus, transgenic plants can be used for the following purposes:

(i) Nutrient quality:

● In section 58.2, under the heading of ‘High-lysine com’, we have described how cereals
rich in certain essential amino acids such as lysine, methionine and tryptophan can be
developed by genetic engineering. Likewise, rice is being modified into Golden rice by
Prof. Inge Potrykus and Dr. Peter Beyer.
● This is done so that vitamin A potential is maintained even after the husks are removed, a
procedure adopted to allow for storage since the husks become rancid. This change may
improve health of millions of people throughout the world.

(ii) Diagnostic and therapeutic proteins:

● Transgenic plants can also produce a variety of proteins used in diagnosis for detecting
human diseases and therapeutics for curing human and animal diseases in large-scale with
low-cost. The monoclonal antibodies, blood plasma proteins, peptide hormones and

35
 cytokinins are being produced in trangenic plants and their parts such as tobacco (in
leaves), potato (in tubers), sugarcane (in stems) and maize (in seed endosperm).

(iii) Edible vaccines:

● Crop plants offer cost-effective bioreactors to express antigens which can be used as edible
vaccines. The genes encoding antigenic proteins can be isolated from the pathogens and
expressed in plants and such transgenic plants or their tissues producing antigens can be
eaten for immunization (edible vaccines).
● The expression of such antigenic proteins in crops such as banana and tomato are useful
for immunization of humans since both of these fruits can be eaten raw. Such edible
vaccines of transgenic plants have the following advantages: lessening of their storage
problems, their easy delivery system by feeding and low cost as compared to the
recombinant vaccines produced by bacteria.

(iv) Biodegradable plastics:

● Transgenic plants can be used as factories to produce polyhydroxy butyrate (PHB,

biodegradable plastics). Genetically engineered Arabidopsis plants produced PHB
globules exclusively in their chloroplasts without effecting plant growth and development.
The large-scale production of PHB may be easily achieved in tree plants such as populus,
where PHB can be extracted from leaves.

Bio-safety measures and regulations for rDNA work

Introduction:

During early era of life sciences, biosafety principles and guidelines were mostly applied
in the field of microbiology and medical practices. In recent time, biosafety guidelines are designed
and applied to research involving recombinant DNA (rDNA) techniques. Handling, production,
storing and transportation of genetically modified organism (GMOs) involve different biosafety
issues under different category. Biosafety practices deal with the application of standard safety
principles handling hazardous material/agents to minimize potential harmful effect on human
health and environment.

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● The definition of biosafety corresponds to recombinant DNA technology can be described
as: “Application of safety principles to laboratory practices in which potentially hazardous
materials or organism are manipulated or handled.”
● National Institutes of Health (NIH) developed certain guidelines on rDNA research in May,
1976. Similarly, Department of Biotechnology (Govt. of India) has also come out with its
set of guidelines which is available at http://dbtindia.nic.in/index.asp . Scope of the
guidelines suggested by DBT encompasses research, large scale operations and
environmental risks. The guidelines prescribe specific actions that include establishing
safety procedures for rDNA research, production and release to the environment and setting
up containment conditions for certain experiments.
● Biosafety regulatory principles and protocols regulates the potential risk and allow access
to the benefits of rDNA technology. Risk assessment and risk management are two
important component of biosafety.
● Biosafety Levels (BSL): All the facilities handling microorganisms and materials
containing recombinant DNA molecules have risk assessment program. Depending on the
risk possessed by the samples, four biosafety levels have been assigned to rDNA research
facilities. Each BSL facility has requirement of unique design features and safety
equipments.
● Biosafety level-I (BSL-I):
○ Agents: Characterized strains of microorganisms known to cause no disease in
healthy adults. eg. E. coli, S. cerevesiae, B. subtilis etc.
○ Recombinant DNA based research activities involving non-pathogenic micro-
organisms for expression of genes using plasmid vectors or low risk viral vectors.
○ Work practice: Standard aseptic microbiological techniques.
○ Safety equipment requirement: Lab coats and eye protection recommended.
○ Facilities: Bench top, sink etc.
● Biosafety level-II (BSL-II):
○ Agents: Handling of micro-organisms which possess moderate hazard to personal
and environment.
○ rDNA based research activities in micro-organisms using non-viral or viral vectors.

37
 ○ Work practice: Standard BSL-I practices with addition of limited access, biohazard
sign, defined procedure for disposal of “Regulated Medical Waste”, proper training
to lab personal and medical surveillance.
○ Safety equipment: Class-II biological safety cabinet, lab coats, gloves, eye/face
protection, physical containment equipment to reduce infectious aerosol exposure
or splashes.
○ Facility: BSL-I facility with addition of autoclave, decontamination facility and
proper airflow.
● Biosafety level-III (BSL-III):
○ Agents: Handling of micro-organisms which are designated as hazardous or
potentially lethal agents to personal and environment.
○ Laboratory personnel must have specific training in handling infectious micro-
organisms and should be supervised by scientist competent in handling infectious
agents.
○ Work practices: BSL-2 practices, with the addition of: controlled access, on-site
decontamination of all waste and lab clothing and medical surveillance.
○ Safety equipment: Class-III biological safety cabinet, lab coats, gloves, eye/face
protection, respiratory protection, physical containment equipment to reduce
infectious aerosol exposure or splashes.
○ Facility: BSL-III facility has specific criteria to meet. Lab should have double door
entry with physical separation of working area from the access corridors,
directional airflow in lab, and no recirculation of exhaust air in the lab, sufficient
decontamination facility, in lab autoclave etc.
● Biosafety level-IV (BSL-IV):
○ Agents: Hazardous and potentially lethal organisms that posses high individual risk
of laboratory transmitted disease for which there is no vaccine or treatment, or a
related agent with unknown risk of transmission.
○ Laboratory personnel must have specialized training in handling BSL-IV agents
and should be supervised by scientist competent in handling infectious agents.
○ Safety equipment: Class-IV biological safety cabinet, lab coats, gloves, eye/face
protection, respiratory protection, physical and containment equipment to reduce
infectious aerosol exposure or splashes.

38
 ○ Facility: BSL-IV facility requires specialized design to minimize the exposure to
risk and only the authorized entry should be permitted in laboratory area in BSL-
IV labs.
● Risk Analysis: The foundation of any safety program is the use of control measures
appropriate for the risk posed by the activities and the agents in use. The process of
analyzing and determining the risk associated with recombinant DNA work is called as
Risk analysis. The principle behind biosafety regulations is to minimize the risk to human
health and safety, and the conservation of environment including safe handling of
hazardous material. Risk analysis consists of three components: risk assessment, risk
management and risk communication. Risk Assessment: Estimation and determination of
risk associated with the handling and production of a recombinant DNA molecule. Risk
Management: The process of analyzing possible prevention measures to minimize the risk
and designing policies accordingly including implementation of them. Risk
Communication: The exchange of information and opinions on risk management between
academic parties, industry, consumers and policy makers.
● Risk Assessment: The biosafety level is determined based on the risk associated with the
work. The principle investigator is responsible for implementing the necessary safety
requirements in his/her laboratory. Risk assessment process accounts the following criteria
to determine biosafety level:
○ i. Pathogenicity – The ability of an organism to cause disease in human system.
○ ii. Virulence – The severity of the disease (lethal/non lethal, availability of cure etc)
in a healthy adult.
○ iii. Proliferation – the subsequent multiplication, genetic reconstruction, growth,
transport, modification and die-off of these micro-organisms in the environment,
including possible transfer of genetic material to other micro- organisms.
○ iv. Transmission route – The possible route of transmission (mucous membrane,
inhalation etc) to establish the disease in human or other organism.
○ v. Infectious dose (ID) – The amount of infectious agent required to cause disease
in healthy human.
○ vi. Antibiotic/disinfectant resistance – The resistance acquired by the infectious
agent to available antibiotic/disinfectant.

39
● The risk associated with recombinant DNA technology can be categorized under different
headings based on their implication on different platforms.
● General Scientific Considerations-
○ A. Characteristics of Donor and Recipient Organisms
■ Taxonomy, identification, source and culture
■ Genetic characteristics of donor and recipient organisms
■ Pathogenic and physiological traits of donor and recipient organisms
○ B. Properties of the modified/engineered organism
○ C. Description of
■ (a) modification,
■ (b) nature, function and source of the insert,
■ (c) vector construction,
■ (d) transfer into host,
■ (e) stability of insert,
■ (f) frequency of mobilization,
■ (g) rate and level of expression, and
■ (h) Influence of the recipient organism on the activity of the foreign protein.
● Human Health Considerations:
A. Characteristics of the modified/engineered organism.
○ Comparison of the recombinant organism to the wildtype organism regarding
pathogenicity.
○ Transmission route to human.
○ Pathogenicity to humans (or to animals if appropriate).

B. Health considerations generally associated with the presence of non-viable

organisms or with the products of rDNA processes. C. Management of personnel exposure,
including biological measures and physical and organizational measures.

● Environmental and Agricultural Considerations:

○ A. Ecological traits relating to the donor and recipient environment.
○ B. Properties of environment where the engineered organism are being applied.

40
 ○ C. Survival, multiplication and dissemination of the engineered organism in the
environment.
○ D. Interactions of engineered organism(s) with biological systems (target and non-
target populations, stability, and routes of dissemination).
○ E. Potential environmental impacts (Effect on target and non-target organisms and
ecosystems).
● Risk Management: Risk management in biosafety issues is related to the target site where
the practice is conducting (laboratory, industry, agriculture field etc.). Recommendations:
General i. Harmonization of approaches to rDNA techniques can be facilitated by
exchanging principles or guidelines for national regulations; developments in risk analysis;
and practical experience in risk management. Therefore, information should be shared as
freely as possible. ii. There is no scientific basis for specific legislation for the
implementation of rDNA techniques and applications. Member countries should examine
their existing oversight and review mechanisms to ensure that adequate review and control
may be applied while avoiding any undue burdens that may hamper technological
developments in this field. iii. Any approach to implement guidelines should not impede
future developments in rDNA techniques. International harmonization should recognize
this need.
● iv. To facilitate data exchange and minimize trade barriers between countries, further
developments such as testing methods, equipment design, and knowledge of microbial
taxonomy should be considered at both national and international levels. Due account
should be taken of ongoing work on standards within international organizations. v. Special
efforts should be made to improve public understanding of the various aspects of rDNA
techniques. vi. For rDNA applications in industry, agriculture and the environment, it will
be important for member countries to watch the development of these techniques. For
certain industrial applications and for environmental and agricultural applications of rDNA
organisms, some countries may wish to have a notification scheme. vii. Recognizing the
need for innovation, it is important to consider appropriate means to protect intellectual
property and confidentiality interests while assuring safety.
● Recommendations: Specific for Industry
○ i. The large-scale industrial application of rDNA techniques wherever possible
should utilize microorganisms that are intrinsically of low risk. Such

41
 microorganisms can be handled under conditions of Good Industrial Large-Scale
Practice (GILSP).
○ ii. If a recombinant microorganism cannot be handled merely by GILSP, measures
of containment corresponding to the risk assessment should be used in addition to
GILSP.
○ iii. Further, research to improve techniques for monitoring and controlling non-
intentional release of rDNA organisms should be encouraged in large-scale
industrial applications requiring physical containment.
● Containment levels: Biosafety containment levels have to be designated for a facility
depending on the level of risk associated with the biological and chemical agents used and
released from it. Following NIH (National Institute of Health, USA) and DBT (Department
of Biotechnology, India) guidelines, different facilities for biological research have been
classified under three containment levels.
● Containment Category 1:
○ Viable organisms should be handled in a production system which physically
separates the process from the environment;
○ Exhaust gases should be treated to minimize (i.e. to reduce to the lowest practicable
level consistent with safety) the release of viable organisms;
○ Sample collection, addition of materials to the system and the transfer of viable
organisms to another system should be done in a manner which minimizes release;
○ Bulk quantities of culture fluids should not be removed from the system unless the
viable organisms have been inactivated by validated means;
○ Effluent from the production facility should be inactivated by validated means prior
to discharge.
● Containment Category 2:
○ Viable organisms should be handled in a production system which physically
separates the process from the environment;
○ Exhaust gases should be treated to prevent the release of viable organisms;
○ Sample collection, addition of materials to a closed system and the transfer of viable
organisms to another closed system should be done in a manner which prevents
release;

42
 ○ Culture fluids should not be removed from the closed system unless the viable
organisms have been inactivated by validated chemical or physical means;
○ Seals should be designed to prevent leakage or should be fully enclosed in
ventilated housings;
○ Closed systems should be located in an area controlled according to the
requirements;
○ Effluent from the production facility should be inactivated by validated chemical
or physical means prior to discharge.
● Containment Category 3: •
○ Viable organisms should be handled in a production system which physically
separates the process from the environment;
○ Exhaust gases should be treated to prevent the release of viable organisms;
○ Sample collection, addition of materials to a closed system and the transfer of viable
organisms to another closed system should be done in a manner which prevents
release.
○ Culture fluids should not be removed from the closed system unless the viable
organisms have been inactivated by validated chemical or physical means; • Seals
should be designed to prevent leakage or should be fully enclosed in ventilated
housings;
○ Production systems should be located within a purpose built controlled area
according to the requirements;
○ Entry should be restricted in the laboratory area and only persons with appropriate
authority should be allowed access to the working area. Effluent from the
production facility should be inactivated by validated chemical or physical means
prior to discharge. Different containment levels have been assigned for rDNA
GILSP (Good industrial large scale practice) micro-organisms. Examples of
containment approaches for recombinant organisms are discussed in the following
slide:
○ Containment levels for different facilities.
○ Recommendations: Specific for Environment and Agriculture

43
■ Considerable data on the effects of different microorganisms on
environmental and human health exist in literature and should be used to
guide risk assessments.
■ It is important to evaluate rDNA organisms for potential risk prior to
applications in agriculture and the environment. However, the development
of general international guidelines governing such applications is premature
at this time. An independent review of potential risks should be conducted
on a case-by-case basis prior to the application.
■ Development of organisms for agricultural or environmental applications
should be conducted in a stepwise fashion, moving, where appropriate, from
the laboratory to the growth chamber and greenhouse, to limited field
testing and finally, to large-scale field testing.
■ Further research to improve the prediction, evaluation and monitoring of the
outcome of applications of rDNA organisms should be encouraged.

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