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RECOMBINANT DNA TECHNOLOGY
RECOMBINANT DNA TECHNOLOGY
• Scientists have developed a number of biochemical
and genetic techniques by which DNA can be
separated, rearranged, and transferred from one cell
to another.
• The discovery of restriction enzymes and many
other enzymes which can be useful as tools in gene
manipulation have brought in vitro recombining of
DNA to a reality.
I. Enzymes commonly used in recombinant
DNA technology
1. Restriction endonucleases (RE)
• Restriction enzymes are found in bacteria.
• Bacteria are always exposed to a danger of being killed by
phage (viruses that infect bacteria).
• In 1972, for the first time RE was used to cut the DNA
specifically and fragments were ligated to first
recombinant molecule.
• Restriction endonucleases are hydrolases and cleave
phosphodiester bonds of double stranded DNA at specific
palindromic sites within the chain to produce 51 PO4 and
31 OH ends.
• They are called as “restriction endonucleases” mainly due
to their natural function in restricting the growth of the
virus that attack bacteria
• There are three types of restriction
endonucleases : Type I, II and III.
• Type I and III recognize specific nonpalindromic
sequences in the DNA chain,
• cleave the chain at different sites away from the
recognition site,
• thus producing DNA fragments of different
length and ends.
• Type II restriction endonucleases, recognize
specific palindromic sequences that range
generally from 4–8 nucleotides and
• cut the chain within the site, thus producing
specific DNA fragments with known ends
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2. DNA polymerase
• DNA polymerases synthesize complementary
nucleotide sequence on a template nucleotide
strand.
• DNA polymerase I, isolated from E. coli, synthesizes
a complementary strand on a template DNA in 51 →
31 direction.
• It also possess low level of exonuclease activity in
both 51 → 31 and 31 → 51 directions.
• This enzyme is used in labeling of DNA to prepare
probe.
• Klewnow enzyme is the large fragment of the DNA
Polymerase I of E. coli.
• It possess 51 → 31polymerase activity and 31 →51
exonuclease activity but lacks the exonuclease activity in
51 → 31 direction.
• Tag DNA polymerase is isolated from a bacterium Thermus
aquaticus, living in hot springs and active even at 94ºC.
• This enzyme is highly thermostable for which it is used for
DNA amplification during polymerase chain Reaction (PCR).
It does not have 31 → 51 exonuclease activity and hence
cannot carry out proof reading
3. Reverse Transcription of mRNA
• Recently, a superior method of selecting desired
genes has been discovered, which is called reverse
transcription of mRNA.
• The m-RNA is mixed with the enzyme “reverse
transcriptase”.
• This enzyme was found in some viruses having RNA
as genetic information instead of DNA.
• The virus utilizes reverse transcriptase to catalyze the
reverse process of synthesizing a complementary
DNA chain on an RNA template, and to insert it into
the chromosome of the host cell
4. DNA ligase
• Two DNA fragments from the same or different sources can
be joined to produce a recombinant DNA.
• DNA ligase performs this function by catalyzing the formation
of phosphodiester bond between adjacent 31 hydroxyl and
51 phosphate termini of the two different DNA fragments.
• DNA ligase produced by T4 bacteriophage is used, which
needs ATP and Mg++ for its activity.
• T4 DNA ligase can ligate (join) both double stranded DNA and
single stranded DNA fragments, DNA fragments having
compatible staggered ends, and those with blunt ends.
• The rate of ligation of compatible staggered ends is 50 – fold
higher than that of blunt ends.
• It repairs nicks in double stranded DNA molecules.
II. VECTORS
• Vectors are the carrier DNAs into which ‘foreign’ DNAs or
genes of interest are spliced to make a recombinant DNA.
• Vectors along with this ‘foreign’ DNAs (i.e. recombinant DNA)
are then introduced into appropriate host cell and are
maintained for study or expression.
• Organisms with chimeric property can be produced with the
help of cloning vectors.
There are two types of vectors.
• cloning vectors and
• expression vectors.
a. Cloning vectors
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Used for obtaining millions of copies of cloned DNA
segment.
The cloned genes in these vectors are not expected
to express themselves at transcription or
translational level.
Cloning vectors are used for creating genomic
library or preparing the probes or genetic
engineering experiments or other basic studies.
Most cloning vectors were originally derived from
naturally occurring extrachromosomal elements
such as bacteriophages and plasmids.
b. Expression vectors
• Allow the expression of cloned gene, to give the
product (protein).
• This can be achieved through the use of promoters
and expression cassettes and regulatory genes
(sequences).
• Expression vectors are used for transformation to
generate transgenic plant, animal or microbe where
cloned gene expresses to give the product.
• Commercial production of product of cloned gene
may also be achieved by high level expression using
the expression vectors.
1. Plasmids
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The occurrence of plasmids in E. coli came to light in the early
1950’s through the pioneering work of Joshua Lederberg in
the USA and William Hayes in England.
Plasmids are genetic elements that are stably inherited
without being a part of the chromosome (s) of their host
cells.
They are found in bacteria and fungi of many kinds but not in
higher eukaryotes and are not essential to the survival of the
host cell.
They may be composed of DNA or RNA and may be linear or
circular.
Double-stranded DNA plasmids appear to exist as
predominantly covalently closed circular molecules in
bacterial cells.
Both circular and linear plasmids are found in yeast and other
fungi.
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Linear yeast plasmids composed of either RNA or DNA, can encode
protein toxins that inhibit the growth of sensitive yeasts.
The number of molecules of a plasmid found in a single bacterial
cell is termed as copy number
The smallest bacterial plasmids are about 1.5 kb and the largest are
greater than 1500 kb. The vast majority are circular.
However, several very large linear DNA plasmids, up to 500 kb long,
have been found in species of Streptomyces and Nocardia.
Smaller plasmids are much desirable for gene cloning experiments.
Larger plasmids are less in number whereas smaller ones are more
in number.
Plasmids with larger copy number are more useful for gene cloning
experiments.
Plasmid PBR 322 is derived from transposon Tn3, plasmid pMBI, and
plasmid pSC 101. pMBI- replicon,Tn3 - ampicillin transposon , pSC
101 – tetracycline resistance region.
2. Bacteriophages as cloning vectors
• Phage has a linear DNA molecule. So a single break
creates two fragments.
• Foreign DNA can be inserted between them and two
fragments can be joined.
• Such phages when undergo lytic cycle in host will produce
more chimeric DNA.
• Wild type lambda phage could accommodate only 2.5 kb
of foreign DNA.
• Phage vectors are restructured by removing nonessential
genes and making vector DNA smaller so that larger insert
can be accommodated in phage head during packing.
• Lambda phage such prepared has one Eco RI site and
accommodates 20-25kb of foreign DNA. They are used for
preparing genomic library of eukaryotes
3. Cosmids
• Cosmids are the novel cloning vectors which possess
properties of both plasmid and phage.
• Cosmids were first developed in 1978 by Barbara Hohn and
John Collins.
• Cosmids contain a cos site of phage (which is essential for
packaging of nucleic acid into protein coat) plus essential
features of plasmid (such as plasmid origin of replication, a
gene for drug resistance) and several unique restriction
sites for insertion of DNA to be cloned.
• Cosmids can be perpetuated in bacteria in plasmid form,
but can be purified by packaging in-vitro into phages.
• Advantage of using cosmid vector is that larger DNA can be
cloned than what is possible with phage of plasmid.
4. Yeast Cloning Vectors
• Yeast cloning vectors are the carrier DNA molecules into yeasts.
• Yeasts are eukaryotes. Yeast artificial chromosome (YACS) are most
sophisticated yeast vectors.
• They have centromeric and telomeric region of a chromosome.
These regions are needed to allow chromosome to be replicated in
yeast cells.
• Due to origin of replication that is present, replication of DNA
occurs.
• These elements are placed in single DNA fragment which can be
used as vector to clone foreign DNA into yeasts.
• The advantages of YAC include very large piece of DNA can be
cloned.
• Only single copy of YAC is present per cell.
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