BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.

,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

LECTURE 32

TOOLS AND TECHNIQUES IN MOLECULAR BIOLOGY-OVERVIEW

The development of recombinant DNA technology (rDNA technology) permitting the transfer of genetic
material between widely divergent species has opened a new era of research into the structure and
function of the genome. The rDNA technology is defined as "the formation of new combinations of
heritable material by the insertion of nucleic acid molecules, produced by whatever means outside the
cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host
organism in which they do not naturally occur but in which they are capable of continued propagation.
The rDNA technology has provided the means to achieve: 1) the fractionation of individual DNA
components of complex genomes, 2) the amplification of cloned genes, 3) the opportunity to study the
expression of individual genes thus cloned and 4) the potential to create new genetic combinations.
There are several other terms that can be used to describe the technology, including gene
manipulation, gene cloning, genetic modification and genetic engineering. The term genetic
engineering is often thought to be rather emotive or trivial, yet it is probably the label that most people
would recognize.

DNA MANIPULATION ENZYMES

Nucleases:
Enzyme nucleases cut or digest the DNA molecule by breaking phosphodiester bond, which is present in
the DNA molecule. Mainly there are two types of nucleases such as exonucleases and endonucleases.
Exonucleases digest the phosphodiester bond which is present at the ends of the DNA molecule.
Endonucleases digest the phosphodiester bonds which are present in the middle of the DNA molecule.

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

Restriction endonuclease enzymes cleave the specific phosphodiester bond present in the DNA
molecule. There are three types of restriction enzymes are there, such as Type-I, Type-II and Type-III.
Restriction enzyme Type-II is used commonly in molecular biotechnology or genetic engineering as they
have the ability to digest phosphodiester bond present in the specific sequence of the DNA. They are
code specific. To do their function they require specific temperature and magnesium concentration.
They produce both blunt end and sticky end after the digestion.

Types of Restriction Endonucleases

The restriction endonucleases can be divided into three groups as type I, II and III. Types I and III
have an ATP dependent restriction activity and a modification activity resident in the same multimeric
protein. Both these types recognize unmethylated recognition sequences in DNA. Type I enzymes cleave
the DNA at random site, whereas Type III cleave at a specific site. Type II restriction modification system
possess separate enzymes for endonuclease and methylase activity and are the most widely used for
genetic manipulation. The properties of three types of restriction endonucleases and a list of enzymes
are given below.

Some restriction endonucleases and their recognition sites

Restriction enzymes that have the same recognition sequences can be isolated from different bacterial
species.

Such enzymes are called isoschizomers.

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

For example, Mbol from Moraxella boris and Sau3A from Staphyhcoccus aureus recognize the sequence
GATC. AatII (recognition sequence: GACGT↓C) and ZraI(recognition sequence: GAC↓GTC) are
neoschizomers

Ligases: Tool for joining DNA fragments

Ligases:
Enzyme ligases help in binding two pieces of DNA by forming phosphodiester bonds. Ligases form two
phosphodiester bonds one at each strand of the DNA molecule or in other words it act as biological glue.
Ligases enzyme has the capacity to bind both blunt end and sticky end produced by restriction
endonuclease enzymes. Adaptors, linkers and homopolymer tailing produce sticky ends.

Ligase mediated joining of nick in the DNA

Polymerases:
Polymerases synthesize a new strand of DNA molecule complementary to the existing DNA or RNA
strand, which acts as template. DNA polymerases require a primer to do their job that is DNA strand
synthesis. DNA polymerase type-I has got both activity like DNA polymerization and also DNA
degradation or nick formation. Klenow fragments are fragments of DNA polymerase type-I enzyme
which has got polymerase activity but lacks the nucleases activity.

DNA Modifying Enzymes:

Alkaline phosphatase:
Enzyme alkaline phosphatase extracted from E. coli or calf intestine degrades phosphate group present
in the 3 prime end of the DNA molecule.
Dephosphorylation of termini: The main function of DNA ligase is to produce a phosphodiester bond
between adjacent nucleotides if one contains a 5' PO4 group and the other a 3' –OH group. Thus,
removal of terminal 5' PO4 groups from the cleaved DNA will prevent self ligation. Dephosphorylation of
termini can be carried out by treating linearised DNA with bacterial alkaline phosphatase. The
dephosphorylated DNA molecules can be religated with phosphorylated passenger DNA to produce
functional recombinant DNA molecules.

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

Terminal deoxy-nucleotidyle transferase:

Terminal deoxy-nucleotidyle transferase enzyme is extracted from calf thymus tissue, which has got the
capacity to add one or more number of nucleotide in 3 prime terminus of the DNA molecule.

Topoisomerases:
Enzyme topoisomerasees change the confirmation of covalently closed circular DNA molecule, such as
plasmids by removing the supercoils present in the circular DNA molecule. Enzyme topoisomerase plays
a very important role during DNA replication but its use in genetic engineering is still not very clear.

Exonucleases:
– Shortening of dsDNA
– Removal of single stranded ends (sticky ends)
– Removal of strands separately:
• Single strands removed by treatment with ExoIII (3’ to 5’ Exo remove
nucleotides from 3’ end not from 5’ end)
• Followed by S1 or mung bean nuclease treatment – shortening of dsDNA
• Protect 3’ end with phosphorothioate nucleotide analogue – prevent action of
ExoIII
– Removal of both strands together:
• Treat the DNA with Bal31 – removes nucleotides – both strands simultaneously
at both ends
• Has 3’ to 5’ exonuclease and an endonuclease activity
• Shortening of dsDNA

Generating DNA fragments using restriction endonucleases

Restriction endonucleases are enzymes that recognize specific sequences within duplex DNA
molecules and cut the DNA at or near these sites. More than 500 different restriction endonucleases
have been discovered. These enzymes can be grouped into three types viz. Type I, II and III. For practical
purposes, the Type I and III restriction enzymes are not much used in rDNA technology. The real
precision scissors are the Type II enzymes. Type II restriction endonucleases recognize and cut DNA
within particular sequences of tetra, penta, hexa or hepta nucleotides which have an axis of rotational
symmetry. In the following examples, different restriction enzymes cut the DNA at specific sequences as

indicated by arrows.

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

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.

VECTORS - PLASMIDS, PHAGEMIDS, COSMIDS, BAC, YAC

DNA Vectors: The carriers of DNA molecules

DNA vectors and their properties

One of the most important elements in gene cloning is the vector, which in conjunction with the
passenger DNA forms the recombinant DNA which can be propagated in suitable host cells. In order to
perform its function, a vector must possess the following properties:
They should be capable of autonomous replication in at least one host organism.
They should be of small size, since this aids the preparation vector DNA and reduces the complexity
of analyzing recombinant molecules.
mplifying the cloned sequence by occurring in multiple copies. High
copy number facilitates in maximizing expression of cloned genes.
There should be a unique cleavage site for a range of restriction endonucleases. Occurrence of
multiple cleavage sites reduces the likelihood of functional recombinant DNA formation.

They should permit detection by simple genetic tests, of the presence of passenger DNA inserted at
cloning site.
They should have appropriate transcriptional and translational signals located adjacent to cloning
sites for better expression of cloned DNA sequences.
They should have host specificity when there is biological containment for a vector.

A variety of different cloning vectors have been developed by using the items mentioned above
as guidelines. They are as follows: plasmids, phages, cosmids, phasmids, shuttle vectors, expression
vectors and single stranded DNA

Plasmids
Plasmids are self-replicating, double stranded, circular DNA molecules that are maintained in
bacteria as independent extra chromosomal entities. These are also found in some yeast but not in
higher eukaryotes. Plasmids are widely distributed throughout the prokaryotes, vary in size from less
6 6
than 1 x 10 to greater than 200 x 10 Da and are generally dispensable.

Plasmids can be grouped into two major types: conjugative and non-conjugative. In conjugative
plasmids transfer genes (tra) and mobilizing genes (mob) are present whereas in non-conjugative
plasmids tra genes absent. The non-conjugative plasmids can be mobilized by another conjugative

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

plasmid present in the same cell, if the mob gene is intact. Non-conjugative differ from conjugative
plasmids by the absence of tra gene.

A list of naturally occurring plasmids and their properties are furnished below.

In general plasmid cloning vectors are designated by a lowercase ‘p’ which stands for plasmid,
and some abbreviations that may be descriptive.

pBR322 plasmid
Plasmid pBR322 is the one of the best studied and most often used ‘”general purpose” plasmids. The BR
of the pBR322 recognizes the work of the researchers F. Bolivar and R. Rodriguez, who created the
plasmid and 322 is a numerical designation that has relevance to these workers. pBR322 is 4362 base
pair long and completely sequenced. pBR322 carries two antibiotic resistance genes. One confers
r r
resistance to ampicillin (Amp ) and the other confers resistance to tetracycline (Tet ) There are eleven
known enzymes which cleave pBR 322 at unique sites. For three of the enzymes, Hind III, Bam HI and Sal
r r
I, the target site lies within the Tet genes and for another two, Pst I and Pru I, they lie in Amp genes.
Thus cloning in pBR 322 with the aid of these enzymes results in insertional inactivation where the
inserted DNA disrupts the function of the gene containing the cloning site. Where the cloning site is
within in an antibiotic resistance gene, such insertional inactivation results in transformants sensitive to
the appropriate antibiotic. Thus, insertional inactivation helps in the selection of recombinants.

General structure of pBR322 plasmids.

Phages
Derivatives of phage have been developed as cloning vectors since the early days of gene
technology. The phage derivatives are considered to be the most suitable cloning vehicles for cloning
genomic eukaryotic DNA because of the following advantages over the plasmids.
 Thousands of phage plaques can be obtained in a single petridish.
 Selection by DNA-DNA hybridisation is possible
 In vitro packaging into empty phage head is possible thus increasing phage infectivity
 Size selection of the packaged DNA is possible

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

 Millions of independently cloned virus particle can be constituted to form a gene library.

Bacteriophage is a genetically complex but very extensively studied virus of E. coli. The DNA of phage, in
the form in which it is isolated from the phage particle is a linear duplex molecule of 48502 bp (~49kb) in
length. The DNA isolated from virus particles is a double stranded linear molecule with short
complementary single stranded projections of 12 nucleotides at its 5’ ends. These cohesive termini also
referred to as cos sites, allow the DNA to be circularized after infection of the host cell.

The genetic map of phage λ comprises approximately 40 genes which are organized in functional
clusters. Genes coding for head and tail are proteins (genes A-J) are on the left of the linear map. The
central region contains genes, such as int, xis, exo etc. which are responsible for lysogenisation i.e the
process leading to the integration of viral DNA and other recombination events. Much of this central
region is not essential for lytic growth. Genes to the right of the central region comprise six regulatory
genes, two genes (O and P) which are essential for DNA replication during lytic growth and two more
genes (S and R) which are required for the lysis of the cellular membranes.
In the phage DNA, larger central region is not essential for phage growth and replication. This
region of phage can be deleted or replaced without seriously impairing the phage growth cycle. Using
this non-essential region of phage λ, several phage vector derivatives have been constructed for efficient
gene cloning.

Cosmids

Plasmids containing phage cos sites are known as cosmids. Cosmids can be used to clone large
fragments of DNA by exploiting the phage in vitro packaging system. Since cosmids have advantages of
both plasmids and phage vectors they can be delivered to the host by the more efficient infection
procedures rather than by transformation. Cloning with cosmid vectors has widened the scope of
plasmid cloning in the following ways.

 The infectivity of plasmid DNA packaged in phage head is at least three orders of magnitude higher
than that of pure plasmids DNA.
 The process almost exclusively yields hybrid clones so that a subsequent selection for recombinant
DNA becomes unnecessary.
 In contrast to normal plasmid transformations, the system strongly selects for clones containing
large DNA inserts. It is therefore, well suited for generating genomic libraries.

General Structure of a cosmid vector

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

The following table provides a list cosmid vectors and their structural features.

Phasmids

Phasmids, also called as phagemids, are hybrids formed between small multicopy plasmids and
bacteriophages. A phasmid can be propagated as a plasmid or lytically as a phage. Lytic functions of
phasmid can be switched off by propagation in the appropriate lysogene where the plasmid origin of
replication is used for maintenance. The phasmid may replicate as phage if propagated in a non-
lysogenic strain. In the case of phasmids based on λ, such as λ1130, the temperature sensitive gene, cI857
carried by the vector may be used to switch between replication modes, simply by growing the host at
the permissive (plasmid mode) or restrictive (phage mode) temperature.

Phasmids are particularly useful in the generation and analysis of mutations exhibiting non-
selectable or lethal phenotypes, such as those affecting the replication of plasmids. Phasmids may also
be used as phage replacement vectors and for directing the high level expression of protein from cloned
sequences by replication in the phage mode.

Shuttle vectors
Shuttle vectors normally comprise an E. coli plasmid or part of such plasmid (e.g., pBR 322),
ligated in vitro to a plasmid or virus replicon from another species. Shuttle vectors can be made, for
example, for E. coli/B. subtilis, E. coli/yeast or E. coli/mammalian cells. The shuttle vector strategy
permits the exploitation of the many manipulative procedures, such as amplification, available in E. coli
(or other genetically well characterized species such as B. subtilis or S. cerevisiae) backgrounds. The
ability to transfer cloned genes across species boundaries is of potential value in the genetic
manipulations of industrially important species and this can be achieved by using shuttle vectors.

Expression vectors

In DNA cloning experiments all the genes cloned are not expressed fully because of weak
promoters in vector DNA. This can be dramatically improved by placing such genes downstream of
strong promoters. An additional problem in maximizing expression of cloned genes in E. coli which is
frequently encountered with genes from a heterologous source is that the gene carries no translation
start signal which can be efficiently recognized by the E. coli translation system. This problem may arise
for heterologous genes cloned into any host. Thus, even though the gene can be transcribed from a
promoter within the vector, the resulting mRNA is poorly translated and little or no protein product will
be synthesized. In such cases alternative strategies available are fusing the gene to amino terminal
region of vector gene that is efficiently translated in the host or coupling the gene to a DNA fragment
carrying both strong promoter and a ribosomal binding site. Vectors with this additional feature are
called expression vectors.

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

Bacterial artificial chromosome (BAC)

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-
plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role
because they contain partition genes that promote the even distribution of plasmids after bacterial cell
division. The bacterial artificial chromosome's usual insert size is 150-350 kbp.

A similar cloning vector called a PAC has also been produced from the bacterial P1-plasmid.
BAC Vector - Common gene components
oriS, repE – F: for plasmid replication and regulation of copy number.
parA and parB – for partitioning F plasmid DNA to daughter cells during division and ensures
stable maintenance of the BAC.
A selectable marker for antibiotic resistance; some BACs also have lacZ at the cloning site for
blue/white selection.
T7 & Sp6 – phage promoters for transcription of inserted genes.

Yeast Artificial Chromosome (YAC)

Yeast Artificial Chromosome (YAC) is a vector used to clone DNA fragments larger than 100 kb and up to
3000 kb. YACs are useful for the physical mapping of complex genomes and for the cloning of large
genes. First described in 1983 by Murray and Szostak, a YAC is an artificially constructed chromosome
that contains a centromere, telomeres and an autonomous replicating sequence (ARS) element, which
are required for replication and preservation of YAC in yeast cells.
Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal
plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to
express eukaryotic proteins that require post-translational modification.

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BTB 103 Molecular Biology (2+1) – 2017 Syllabus Dr. S. RAJESH, PhD.,
Assistant Professor (Biotech.)
Centre for Plant Molecular Biology & Biotechnology
TNAU, Coimbatore – 641 003.

A plasmid-derived origin of replication (ori) and an antibiotic resistance gene allow the YAC vector to be
amplified and selected for in E. coli.
TRP1 and URA3 genes are included in the YAC vector to provide a selection system for identifying
transformed yeast cells

Host systems for cloned vectors

Escherichia coli
Vectors and their hosts form integrated system for constructing and maintaining recombinant
DNA molecules. The choice of a particular host -vector system depends on a variety of factors, including
ease and safety of manipulations and the likelihood of expression of cloned genes. Among the host
system E. coli system remains well exploited one. strains that are considerable less disabled and hence
more easily handled than other hosts.

Bacillus subtilis
Bacillus subtilis is the best characterized of all Gram positive bacteria. It has a well-defined
genetic map and efficient systems for transformation and transfection. In addition, B subtilis is
commercially important since procedures for the synthesis of peptide antibiotic and extracellular
enzymes, such as proteases are made available. Further, the species is nonpathogenic which makes it a
safe host for cloning potentially hazardous genes. However, B. subtilis does sporulate readily, thus
increasing the probability that cloned genes would survive outside the laboratory or fermenter.

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