• Biotechnology is the use of biology to develop new products, methods, and organisms

which can be used systematically to improve human health and society. The concept of
biotechnology started long ago with the domestication of plants, and animals and the
discovery of fermentation.
• Biotechnology is the branch of science which deals with the application of biological
techniques for human welfare. It involves the modification of genes by the enzymatic
action which leads to the production of some biological entity like antibodies, vaccines etc
which benefits the mankind.
• According to the definition of the European Federation for Biotechnology «biotechnology
is an integrated application of natural and engineering sciences with the aim of using living
organisms, cells and their component parts for products andservices» (EFB,1989).
• Today, the five branches into which modern biotechnology is divided — human,
environmental, industrial, animal and plant — help us fight hunger and disease,
• Károly Ereky (German: Karl Ereky; 20 October 1878 – 17 June 1952) was a Hungarian
agricultural engineer. The term ‘biotechnology’ was coined by him in 1919. He is regarded
by some as the “father” of biotechnology.
• Pushpa Mittra Bhargava is considered to be the Father of Indian Biotechnology
Principles of Biotechnology
According to modern Biotechnology, the main principles of biotechnology are:
(i) Genetic engineering:
The methods to alter the chemistry of the genetic material (DNA & RNA) to introduce genes into host
species and thus alter the phenotype of the host organism.
• In simple words, genetic engineering can be described as the manual addition of a new DNA
into an organism.
• It aids the addition of such traits that are not originally found in the organisms.
• Recombinant DNA is required to create Genetically Modified Organisms (GMO.)
• An area of chromosome (gene) is spliced.
• Genetic disorders in humans can be corrected using genetic engineering.
• Selective breeding has been in the world since ancient times.
❖ Jack Williamson used the word ‘Genetic Engineering’ in his science fiction novel Dragon’s
Island which was published in 1951.
❖ First recombinant DNA molecules were created by an American Biochemist, Paul Berg.
Tools of genetic engineering
Restriction enzymes: restriction enzymes are one of the most important tools in genetic engineering;
they are also called restriction endonucleases. These are important proteins produced by micro bacteria
that cut DNA at particular sites along with the molecule. Eliminating infecting organisms is the main
objective of the restriction enzyme in a bacterial cell.
Electroporation: insertion of selected genes in specially cultured cells or tissues (commonly used for
bacteria, yeast and protoplasts) using the electric pulses will create pores in the membrane of the cell
which will allow the flow of selected genes; this is called electroporation.
DNA ligase: DNA ligase is called the biological glue which plays a great role in DNA repair and DNA
replication. In this process, DNA ligase is used to generate rDNA. As we discussed earlier, restriction
enzymes will cut, forming uneven strands, and DNA ligase helps connect them together.
Genetic vectors: vectors are also called genetic vehicles. As the name suggests, the vectors help
transport the marked and selected gene to the assigned gene’s location. The ability to replicate fast is
one of the specialities of biological vectors. Artificial chromosomes, viruses and plasmids can be used
as vectors.
Other common tools include Gel electrophoresis, other enzymes, RNA, transgene, plasmid, PCR,
etc.
Applications of genetic engineering:
• Agriculture
• Medicine
• Health Science
• Crime Scene
• Animal Husbandry
• Molecular Biology
• Gene Therapy
• Production of Hormones
• Production of Antibodies
• Model Animals
• Model Crops
(ii) Bioprocess engineering:
This process involves the maintenance of a sterile atmosphere in chemical engineering processes to
allow the growth of only the desired microbes in significant quantities to manufacture biotechnological
products like antibiotics, vaccines, etc.

Recombinant DNA:
• Traditional hybridisation procedures often lead to the multiplication of both
unwanted and desired genes. At the same time, the new strategies of
recombinant DNA technology have brought a breakthrough in this limitation
with the help of gene cloning and gene transfer.
• Recombinant DNA technology introduces only the desired genes without
adding undesirable genes to the target organism.
• DNA will only multiply in the progeny cells when incorporated into the
recipient’s genome and inherited with the host DNA. The alien piece of DNA
becomes part of a chromosome capable of replication.
• A specific DNA sequence in a chromosome responsible for starting
replication is called the ‘Origin of Replication’.
• To multiply a foreign piece of DNA in an organism, it needs to be a part of a
chromosome with the ‘Origin of Replication’. The ori permits the foreign piece of
DNA to replicate and multiply in the host organism by a process called cloning..
The construction of the first recombinant DNA:
 • The first artificial recombinant DNA emerged from the potential of linking a
gene encoded antibiotic resistance with a plasmid of Salmonella
typhimurium. Stanley Cohen and Herbert Boyer achieved this by isolating the
antibiotic resistance gene in 1972. They cut out a piece of DNA from a plasmid
responsible for contributing to antibiotic resistance.
• Restriction enzymes cut the DNA at specific locations and link the cut piece
of DNA with the plasmid DNA. These plasmid DNA behave as vectors to
transfer the piece of DNA adhered to it.
• With the aid of the enzyme DNA ligase, the antibiotic resistance gene was
linked with the plasmid vector.
• The new combination of circular autonomously replicating DNA created in
vitro is called recombinant DNA.
• When this DNA is transferred into Escherichia coli, it could replicate using the
new host’s DNA polymerase enzyme and produce multiple copies.
• The cloning of antibiotic resistance genes in Escherichia coli was the ability to
multiply copies of antibiotic resistance genes.
The three basic steps in genetically modifying an organism:
1. Identification of DNA that comprises suitable genes
2. Desirable DNA is introduced into the host organism.
3. After maintaining introduced DNA in the host organism, the DNA is transferred to its
progeny.
Tools of Recombinant DNA Technology :
The critical tools that are involved in genetic engineering:
• Restriction enzymes
• Polymerase enzymes
• Ligases
• Vectors
• Host organism
Restriction Enzymes:
• Two enzymes in Escherichia coli were isolated. They were responsible for
restricting the growth of bacteriophages. One of these added methyl groups
to DNA while the other cut DNA. This was known as restriction endonuclease.
• Hind II was the first restriction of the endonuclease. Its functioning depended
on a specific DNA nucleotide sequence that was isolated and characterised
after five years.
 • Hind II cut DNA molecules at specific points by recognising a specific
sequence of six base pairs. This specific base sequence is termed the
recognition sequence for Hind II.
• However, today, over 900 restriction enzymes have been secluded from more
than 230 strains of bacteria, each of which recognises various recognition
sequences.
• NOMENCLATURE: While naming these enzymes, the first letter of the name is from
the genus. The second two letters are derived from the species of the prokaryotic
cell from which they were isolated. For Example, EcoRI (Escherichia coli RY 13).
• The letter ‘R’ is derived from the strain’s name. Roman numbers that follow
the names indicate the order of isolation of the enzymes from that strain of
bacteria.
• Restriction enzymes belong to a larger class of enzymes called nucleases.
These are of two kinds:
1. Exonucleases: get rid of nucleotides from the ends of the DNA.
2. Endonucleases: make cuts at certain positions within the DNA.

• A restriction endonuclease operates by ‘inspecting’ the length of a DNA

sequence. Once the specific recognition sequence is found, it will combine
with the DNA.
• Then each of the two strands of the double helix is cut at distinct points in
their sugar-phosphate backbones.
• Each restriction endonuclease will distinguish specific palindromic
nucleotide sequences in the DNA.

What are Palindromes?

Palindromes are groups of letters that form the exact words when read both forward and backward, e.g.,
MALAYALAM Racecar , rotator. The palindrome in DNA is a sequence of base pairs. It reads the
same on the two strands when the orientation of the reading is kept the same.
For Example, the following sequences are read-alike on the two strands in the 5′ to 3′ direction. This is
also true if the arrangement is read in the 3′ to 5′ direction.
5′ —— GAATTC —— 3′
3′ —— CTTAAG —— 5′
Restriction enzymes cut up the DNA strand between the same two bases on the opposite strand and a
little away from the centre of the palindrome sites. This leaves portions of single-stranded DNA at the
ends. Overhanging stretches called sticky ends, present on each strand, form hydrogen bonds with
complementary cut counterparts. This stickiness of the ends enables the action of the enzyme DNA
ligase.
In genetic engineering, ‘recombinant’ molecules of DNA are formed with the help of restriction
endonucleases composed of different DNA genomes. When the same restriction enzyme cuts the DNA,
the DNA fragments formed have the same kind of ‘sticky-ends’. These are joined together using DNA
ligases
Separation and isolation of DNA fragments:
After cutting DNA by a restriction endonuclease, the fragments get separated by gel electrophoresis.
DNA fragments being negatively charged molecules are separated by moving towards the anode in the
presence of an electric field through a medium. The most commonly used medium is agarose gel (a
natural polymer) extracted from seaweeds. The separation of DNA fragments is based on their size
through the sieving effect given by the agarose gel. Thus, the smaller fragments move farther on the
agarose gel.
The separated DNA fragments can be seen only after staining the DNA. A compound, ethidium
bromide, is used for visualisation, followed by exposure to UV radiation. You will observe bright
orange-coloured bands of DNA when exposed to UV light. The separated DNA bands are extracted
from the gel piece after being cut. This process is called elution. The purified DNA fragments are used
to construct recombinant DNA by joining them with cloning vectors.
Cloning Vectors:
• Because of their more significant number per cell, Bacteriophages have
large copy numbers of their genome within the bacterial cells.
• Some plasmids only consist of one or two copies per cell, while others may
have 15-100 copies per cell.
• Multiplication of equal numbers to the copy number of the plasmid becomes
easy with the ability to link an alien piece of DNA.
• Vectors are engineered to help the easy linking of foreign DNA and the
selection of recombinants from non-recombinants.

Following features are required to facilitate cloning into a vector:

1. Origin of replication (ori): ori is a sequence from where replication begins.
Any piece of DNA linked to this sequence, further replicates within the cells of
 a host. This sequence is also responsible for controlling the copy number of
the linked DNA. Thus, if anyone wants to retrieve a large number of copies of
the target DNA, it should be cloned in a vector whose origin supports a high
copy number.
2. Selectable marker: Apart from ‘ori’, the vector requires a selectable marker.
The selectable marker assists in the identification and elimination of non-
transformants. Also, it selectively allows the growth of the transformants.
Transformation is the procedure through which a piece of DNA is introduced
into a host bacterium. The genes encode antibiotic resistance. These include
ampicillin, chloramphenicol, tetracycline or kanamycin, etc., which are useful
selectable markers for E. coli.
3. Cloning sites: The vector requires very few single recognition sites for the
restriction enzymes to link foreign DNA. The availability of recognition sites within
the vector will produce numerous fragments, which will cause complications in
gene cloning.
The ligation of foreign DNA is carried out at a restriction site that occurs in one of the two antibiotic
resistance genes. For instance, you can ligate an alien DNA at the BamH I site of the tetracycline
resistance gene in the vector pBR322.

The transformants growing on an ampicillin containing matrix are then transported on a tetracycline

matrix. The recombinants will multiply in an ampicillin containing matrix but not on that containing
tetracycline. Non- recombinants will grow on the medium with both antibiotics. In this case, only one
antibiotic resistance gene helps select the transformants. In contrast, the other antibiotic resistance gene
gets ‘inactivated due to insertion’ of foreign DNA and helps choose recombinants.
1. Vectors for cloning genes in plants and animals: The methods of transferring
genes into plants and animals extracted from bacteria and viruses have
been known for ages. For instance, Agrobacterium tumefaciens is a
pathogen of various dicot plants which can deliver a piece of DNA known as
‘T-DNA’. T-DNA converts normal plant cells into a tumour and directs these
 tumour cells to produce the chemicals required by the pathogen. In the
same way, retroviruses in animals can transform normal cells into cancerous
cells.
The tumour-inducing (Ti) plasmid of Agrobacterium tumefacient has now been modified into a cloning
vector that is not pathogenic to the plants but can still use the mechanisms to deliver desired genes into
a variety of plants.

Competent Host:

• DNA is a hydrophilic molecule. Thus, it cannot pass through cell membranes.

In contrast, bacterial cells are made ‘competent’ to accept plasmid DNA.
• The bacteria are treated with a specific divalent cation concentration, such
as calcium. Calcium raises the efficiency with which DNA enters the
bacterium through the pores of the cell wall.
• Recombinant DNA is forced into such cells by incubating the cells with
recombinant DNA on ice.
 • After that, the cells are placed shortly at 42 degrees celsius to give heat
shock and then put back on ice. This allows the bacteria to take up the
recombinant DNA.
• Another way to introduce foreign DNA into a host cell is through micro-
injection. Recombinant DNA is injected directly into the nucleus of an animal
cell.
• Another method appropriate for plant cells is bombardment with high-
velocity micro-particles of gold or tungsten coated with DNA. This procedure
is known as biolistics or gene gun.
• The last method uses ‘disarmed pathogen’ vectors. The vectors transport the
recombinant DNA into the host cell when allowed to infect the cell.

Processes Of Recombinant DNA Technology

The process is explained in detail in our Class 12 Biology Chapter 11 Notes. Below are the summary
points highlighting the key steps in Recombinant DA technology:

Isolation of DNA:
• DNA and other macro_molecules such as RNA proteins and lipids are
enclosed within the cell membrane.
• The cell is treated with enzymes such as lysozyme(bacteria), cellulase(plant
cells) and Chitinase(fungus) that isolate the DNA.
• The treatment with ribbon nucleus proteins can extract RNA through the
treatment with proteins.
• DNA finally precipitates upon adding chill ethanol in a suspension of fine
threads.

Cutting of DNA at Specific Locations:

• Restriction enzyme digestion is performed by incubating purified DNA
molecules with the restriction enzyme.
• The specific enzymes require optimum conditions.
• DNA is negatively charged and moves towards the anode when the Agarose
gel electrophoresis is employed.
• The process is repeated with vector DNA as well.
• The cut source of DNA, the vector DNA with a specific restriction enzyme, the
cut-out gene of interest and the cut vector with space are mixed, followed
by the addition of ligase.
• By this Recombinant DNA is prepared.

Amplification of Gene of Interest using PCR:

• In Polymerase Chain Reaction(PCR), multiple copies of DNA of interest are
synthesised in vitro with the help of two sets of primers; chemically
synthesied oligonucleaotides and theDNA polymerase enzyme.
• DNA polymerase enzymes extend the primers utilising nucleotides provided
in the reaction and the genomic DNA as template.
 • When the DNA replication is repeated many times, this segment of DNA can
be amplified to construct a billion copies.
• Thermostable DNA polymerase achieves repeated amplification and
remains active during high temperature-induced denaturation of double
standard DNA.
• Amplified fragments can be used for ligation with a vector to continue
cloning.

Each cycle has three steps:

(i) Denaturation;
(ii) Primer annealing
(iii) Extension of primers

Insertion of Recombinant DNA into the Host Cell:

• Competent recipient cells take up DNA present in their surroundings.
• If recombinant DNA consists of a gene for resistance to an antibiotic is
transferred into E.coli cells , the host cells transform into ampicillin-resistant
cells.
• Only transformants will grow if they are spread on agar plates containing
ampicillin, while untransformed recipient cells will die.
• The presence of the ampicillin resistance gene allows the selection of the
desired transformed cell.
• The ampicillin resistance gene is called a selectable marker.

Obtaining the Foreign Gene Product:

• The ultimate aim is to produce a desirable protein.
• Knowledge of many technical details is essential when the expression of
foreign genes in host cells is involved.
• The cloned gene of interest is optimised to induce the expense expression of
the protein. This production is taken on a large scale.
• A recombinant protein is expressed in a heterologous host.
• Cells with the cloned gene of interest are grown in a lab for small-scale
production.
• The cultures extract the required protein and then purify it using different
separation techniques.
• With the help of a continuous culture system, the cells are multiplied where
the used medium is drained out, and the fresh medium is added to maintain
 the exponential phase. This culture method produces larger biomass, which
yields a more significant number of desired proteins.

What are Bioreactors?

As
The bioreactor is a large vessel where the different cells such as human or plant, or animal cells can
be cultured to obtain new biological products. It provides optimum conditions like temperature, pH,
substrate, oxygen, etc required for the culturing of cells producing desired products.
(b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged

Downstream Processing:

Before marketing a finished product, it must be subjected to several processes. The processes are:
• Separation and Purification(Collectively referred to as Downstream
processing).
• Formulation with suitable preservatives. In the case of drugs, the Formulation
has to undergo a full clinical trial.
• Proper quality control testing for each product is mandatory.
• The downstream processing and quality control are different for every
product.

Q.1 What happens to a DNA fragment that is transferred into an alien organism?
Ans
Two situations arise:
a. The alien DNA doesn’t integrate to the host’s genome and is lost after the cell containing the alien
DNA dies.
b. The alien DNA integrates to the host’s genome and multiplies as the cell reproduces.
Q.2 What is insertional inactivation? Give an example.
Ans
The inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence is
called insertional inactivation. Any products from the inactivated gene will not work because of the
codes added to it. Cromogenic selectable markers show insertional inactivation. A recombinant DNA

is inserted within the coding sequence of an enzyme, -galactosidase inactivating the enzyme.
The presence of a substrate gives blue coloured colonies if the plasmid in the bacteria does not have

an insert. Presence of insert results into insertional inactivation of the -galactosidase and the
colonies do not produce any colour.
Q.3 Define Biotechnology.
Ans
Biotechnology is defined as the industrial use of living organisms (or their parts) to produce various
products and services. It is the fusion of biology and technology.
Q.4 Expand EFB. What is the definition of Biotechnology according to EFB?
Ans
EFB stands for European Federation of Biotechnology The definition of Biotechnology as given by
EFB: ‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues
for products and services’.
Q.5 What are the two core techniques that enabled birth of modern biotechnology?
Ans
The two core techniques are:
a. Genetic engineering: The ability to alter genetic material of an organism according to ones need
and get the desired phenotypic expression
b. Sterile microbial culturing techniques: The ability to grow desired organism in a contamination
free environment
Q.6 What do you understand by the term genetic engineering? What are the basic three steps
involved in genetic modification of an organism?
Ans
Genetic engineering is the scientific alteration of genes or genetic material to produce desirable new
traits in organisms or to eliminate undesirable ones. It aims at introducing new characteristics or
attributes physiologically or physically, such as introducing a novel trait, enhancing existing ones, or
producing a new protein. It involves:
a. Isolation of DNA
b. Manipulation of the DNA
c. Reintroduction of DNA into cells/organisms.
Q.7 What is the difference between modern biotechnology and biotechnology in general?
Ans
Modern biotechnology involves highly sophisticated techniques such as genetic engineering, protein
and enzyme engineering and in vitro fertilisation and reproduction techniques whereas biotechnology
in general also includes non-sophisticated techniques like agriculture, genetic breeding, curd and
bread making etc. In brief, modern biotechnology is biotechnology at the molecular level.
Q.8 What is the benefit of sexual reproduction over asexual reproduction?
Ans
Sexual reproduction provides opportunities for variations in the genetic makeup of an organism,
which may be beneficial to the organism as well as the population, especially under environmental
stress.
Q.9 Traditional hybridization procedures used in plants and animal breeding, very often lead to
inclusion and multiplication of undesirable genes. How can you overcome this problem?
Ans
We can overcome the problem by the use of genetic engineering where we can isolate and introduce a
single or a set of specific genes without introducing undesired genes into the target organism.
Q.10 What is origin of replication?
Ans
The origin of replication is a particular DNA sequence in the chromosome or a plasmid at which
DNA replication is initiated.
Q.11 What is cloning?
Ans
Cloning collectively refers to the process used to create copies of DNA fragments (molecular
cloning), cells (cell cloning), or organisms. The term also encompasses situations whereby organisms
reproduce asexually.
Q.12 What are ‘molecular scissors’?
Ans
Molecular scissors are enzymes that cut molecules at a specific point.
– Restriction enzymes are molecular scissors that cut double-stranded DNA.
– Ribozymes are molecular scissors that cut RNA.
Molecular scissors are used as tools of modern biotechnology for cutting the desired fragment of
DNA, RNA etc.
Q.13 What are vectors? Write a short note on cloning vectors.
Ans
A vector is an organism that does not cause disease itself but which acts as a vehicle for the disease
causing pathogens. A cloning vector is a small DNA vehicle that carries a foreign DNA fragment. It
is used to transfer the desired gene into the host during genetic engineering experiments. The
insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign
DNA with the same restriction enzyme, then ligating the fragments together. There are many different
types of cloning vectors.
Eg.: Plasmids, Bacteriophages (λ phage), Cosmid, Bacterial Artificial Chromosome (BAC), Yeast
Artificial Chromosome (YAC) etc.

Plasmid pBR322 is an E. coli cloning vector, showing restriction sites

(HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI), ori and antibiotic resistance genes (ampR and
tetR).
Q.14 Write a short note on DNA ligase.
Ans
DNA ligase is a type of enzyme that joins double stranded DNA fragments. DNA ligase is used in
vivo in DNA repair and DNA replication and in vitro in recombinant DNA technologies. DNA ligase
forms covalent phosphodiester bonds between 3′ hydroxyl ends of one nucleotide and the 5′
phosphate end of the another.
Q.15 Why are plasmids and bacteriophages used as cloning vectors?
Ans
It is so because:
• Plasmids and bacteriophages have the ability to replicate inside bacterial
cells without being controlled by the chromosomal DNA.
• Bacteriophages and many plasmids are present in high number of copies
per cell and thus high number of the engineered gene can be obtained.
Q.16 What are the key tools required for genetic engineering?
Ans
The key tools required for genetic engineering are:
a. Enzymes: Restriction endonuclease, DNA ligase, DNA polymerase etc.
b. Vectors: Plasmids, phasemids, cosmids, BACs, YACs etc.
c. Transformation tools like gene gun, microinjection etc.
Other tools required are those used for gene isolation, separation and screening of recombinants.
Q.17 Write a short note on the followings:
a. Ori
b. Selectable marker
c. Cloning sites
Ans
a. Ori: Ori or origin of replication is a particular DNA sequence at which DNA replication is
initiated. The ori binds the pre-replication complex, a protein complex that recognizes, unwinds, and
begins to copy DNA. Prokaryotes have single origin of replication per circular chromosome while
eukaryotes have multiple origin of replication per chromosome (to help speedup the replication of the
usually large genetic content).
b. Selectable marker: Selectable markers are genes coding for a certain known phenotypic
characteristics that helps in identifying and eliminating non-transformed organisms and selectively
permitting the growth of the transformed (having the recombinant gene) organisms. Genes that code
for resistance to antibiotic such as ampicillin, chloramphenicol, tetracycline or kanamycin or impart
specific colours to the organism/ microbial colonies are used as selectable markers.
c. Cloning sites: Cloning site is that region of a vector in which the gene to be transferred to the host
is inserted. The site contains recognition sequences specific to various restriction endonuclease so
that the gene-containing DNA fragment can be inserted into the vector. Multiple recognition
sequences may be present but generally one is preferred to keep thing simple.
Q.18 From which organism restriction endonuclease was first extracted?
Ans
Escherichia coli
Q.19 Write a short note on Agrobacterium tumifaciens.
Ans
Agrobacterium tumifaciens is a rod shaped, gram-negative bacteria that is able to deliver a piece of its
DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to
produce the chemicals required by it. This tumor inducing (Ti) plasmid has been modified into a
cloning vector which can deliver genes into a variety of plants without causing the disease.
Q.20 What is known as recognition sequence of a restriction endonuclease? Give an example.
Ans
Many restriction endonucleases exhibit binding specificity and function only after binding to a
specific DNA sequence. This specific base sequence is known as the recognition sequence for that
particular restriction endonuclease.
Enzyme Source Recognition Sequence Cut
EcoRI Escherichia 5’GAATTC 5’-G
coli 3’CTTAAG AATTC-
3’
3’-
CTTAA
G-5’
HindIII Haemophillus 5’AAGCTT 5’-A
influenzae 3’TTCGAA AGCTT-
3’
 3’-
TTCGA
A-5’
Q.21 Describe the convention for naming restriction endonucleases.
Ans
Naming of restriction enzymes is based on the bacteria from which they are isolated in the following
manner:-

• The first letter of the name comes from the genus.

• The second two letters come from the species.
• The next letter comes from the strain.
• Roman numbers following the names indicate the order in which the
enzymes were isolated from that strain of bacteria.
E.g., Naming of EcoRI

E Escherichia genus

co coli species

R RY 13 strain

I First identified order of identification

in the bacterium

Q.22 What are nucleases? What is the difference between endonucleases and exonucleases?
Ans
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide
subunits of nucleic acids. Restriction endonuclease cleaves the double-stranded DNA at a specific
recognition site after binding to it at that point whereas restriction exonuclease removes nucleotides
sequentially from the end of the DNA.
Q.23 Explain the working of a restriction endonuclease.
Ans
A restriction endonuclease functions by “scanning” the length of a DNA molecule. Once it finds its
specific recognition sequence it binds to the DNA molecule and makes one cut in each of the two
sugar-phosphate backbones of the double helix at the specific points.
Q.24 What is a palindromic sequence? What is its significance in genetic engineering?
Ans
A palindromic sequence is a sequence that has the property of reading the same in either direction.
Restriction endonucleases used in genetic engineering to cut double-stranded DNA have various
palindromic sequences as their recognition sequence. The endonucleases identify these specific
palindromic sequences, binds and cleaves the DNA at that point.
 5’-GAATTC-3’ 5'-G AATTC-3' Action of EcoRI
3’-CTTAAG-5’ 3'-CTTAA G-5' Q.25 What are
sticky ends? What
is its significance?
Ans
A sticky end also called overhangs which is a stretch of unpaired nucleotides at the end of a double-
stranded DNA molecule. These unpaired nucleotides can be in either strand, creating either 3′ or 5′
overhangs.
Sticky ends have the ability to form hydrogen bonds with their complementary cut counterparts and
facilitate the action of DNA ligases.
5′—G AATTC—3′
3′—CTTAA G—5′
Q.26 How is a DNA fragment amplified?
Ans
A DNA fragment is amplified either by polymerase chain reaction or by microbial cloning method.
Q.27 What is the sole purpose of recombinant DNA technology?
Ans
The sole purpose of the recombinant DNA technology is to express a certain phenotypic character or
a protein in an organism in which it is not naturally found. This, in turn, has many applications like in
development of better varieties of animals; pest resistant, drought resistant, high yielding plants; drug
production and gene therapy etc.
Q.28 With a diagram explain the steps involved in recombinant DNA technology.
Ans
The steps involved in recombinant DNA technology are:
i. Identification and isolation of desired gene.
ii. Selection of a vector and inserting the DNA fragment containing the desired
gene into it. This is done with the help of restriction endonuclease and DNA
ligase enzymes.
iii. Inserting the engineered vector into the host cell/organism. This is done by
the use of transformation techniques like biolistic (use of gene gun),
microinjection or disarmed pathogens.
iv. Screening of recombinant organisms by exploiting the selectable marker
introduced along with the desired gene.
v. Maintenance and multiplication of the recombinants obtained.
Steps involved in recombinant DNA technology
Q.29 How do you separate DNA fragments?
Ans
DNA is negatively charged and can be separated using gel electrophoresis. Here, the negatively
charged DNA fragments are forced to move towards the positively charged anode under an electric
field through a matrix, generally agarose gel. The fragments separate according to their size, the
smallest moving the fastest and reaching the fastest from the point of loading.
Q.30 What is agarose?
Ans
Agarose is a polysaccharide extracted from seaweeds and is a constituent of agar. It is widely used as
a medium of gel-electrophoresis.
Q.31 Write a short note on gel-electrophoresis?
Ans
Gel-electrophoresis is a technique for separating a mixture of molecules on the basis of their size
under an electric field. DNA, RNA and proteins can be separated using this procedure. The larger
molecules takes more time to pass through the matrix, generally made of agarose, compared to the
smaller molecules. It can be used both for analysis and purification (small scale) of samples.
Q.32 What is an antibiotic resistance gene? What is its significance in recombinant DNA
technology?
Ans
Antibiotic resistance gene is a gene which is expressed in a microorganism. It is able to withstand the
effect of that specific antibiotic. In recombinant DNA technology, antibiotic resistant genes are used
in the vectors as selectable markers to identify transformed hosts.
Q.33 Define Transformation.
Ans
Transformation is defined as the genetic alteration of a cell resulting from the uptake and expression
of foreign genetic material.
Q.34 What are the various methods available for inserting an engineered vector into the host?
Ans
The various methods are:
a. Use of competent cells: These have the ability to take up extracellular DNA. Competence can be
induced by treating the cells with divalents like calcium and subjecting them to heat shocks.
b. Microinjection: Use of a micro-needle to insert the vector into the cell.
c. Gene Gun: Biolistic particle delivery system injects the vector (coated onto a gold particle) into
the host cell, generally plant cells.
d. Use of disarmed pathogens: Pathogens which are devoid of the virulent gene but retain the ability
to transfer the recombinant DNA into the host.
Q.35 What do you understand by the term competent host with reference to recombinant DNA
technology?
Ans
A competent host is a cell that has the ability to take up extracellular DNA from the environment. In
recombinant DNA technology, competent microbial cells are used in order to avoid expensive and
complex transformation techniques like biolistics and microinjection.
Q.36 Write short note on the following:
a. Micro-injection
b. Biolistics
c. Disarmed pathogen
Ans
a. Micro-injection: Microinjection is a transformation technique where engineered vectors/genes are
injected into the host cell using a micro-needle. The process is carried out under an optical
microscope called the micromanipulator and the DNA molecules can not only be delivered inside the
cell membrane but also the nuclear envelope when needed.
b. Biolistics: Biolistics is a transformation technique involving the use of a gene-gun or the Biolistic
Particle Delivery System. Generally used to transform plant cells, the system uses a heavy metal
particle coated with the engineered vector. This coated particle is fired into the host cells using a
specialized airgun. Specialized biolistics can not only transform genetic material but also cell
organelles.
c. Disarmed pathogen: Pathogens/viruses that are devoid of their virulent genes are called disarmed
pathogens. These pathogen vectors when allowed to infect a host are able to transfer the recombinant
DNA into the cell without causing any disease. Such vectors are however not completely safe as there
are chances of such vectors reverting to virulent forms.
Q.37 Expand PCR. Explain the steps involved in the technique. What is the PCR machine
called?
Ans
Polymerase Chain Reaction.
The steps involved are: – A PCR starts with a denaturing step. The DNA sample is heated to 94-960C
to break the hydrogen bonds between the bases of the two strands.
– When the two strands separate out, the temperature is lowered to 50-560C, which allows the primers
(short DNA fragments that acts as starting point for replication) to anneal (base pair) with the
denatured strands.
– The temperature is raised to 720C at which the Taq polymerase (a thermostable polymerase)
elongates the primer by adding nucleotides.
The PCR machine is called a thermocycler.
Q.38 What is a primer? Why is it required?
Ans
A primer is a short DNA sequence that pairs to a larger DNA fragment at a complementary sequence
and acts as a starting point for DNA replication. DNA polymerase elongates a sequence by adding
nucleotides to it and thus requires a primer strand.
Q.39 What is a recombinant protein?
Ans
A recombinant protein is a protein expressed artificially in an organism with the help of recombinant
DNA technology, e.g., artificial human insulin expressed in E. coli.
Q.40 What is a bioreactor?
Ans
A bioreactor is a device, natural or artificial (bioreactor vessels) that supports biologically active
environment and in which chemical reactions involving organisms and/or biologically active
substances are carried out.
Q.41 Write a short note on pBR322.
Ans
pBR322 is a commonly used E. coli plasmid cloning vector. The molecule is a double-stranded
circular DNA, 4361 base pairs in length. It was the first artificial plasmid created by Bolivar and
Rodriguez and was named after them.