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It occurs in following stages
Generation of DNA fragments & selection of
the desired piece of DNA (e.g. a human gene).
Insertion of the selected DNA into a cloning
vector (e.g. a plasmid) to create a
recombinant DNA or chimeric DNA.
Introduction of the recombinant vectors into
host cells (e.g. bacteria).
Multiplication & selection of clones
containing the recombinant molecules.
Expression of the gene to produce the
desired product.
1. Molecular tools of genetic engineering.
2. Host cells-the factories of cloning.
3. Vectors-the cloning vehicles.
4. Methods of gene transfer.
5. Gene cloning strategies.
Restriction endonucleases - DNA cutting
enzymes:
Restriction endonucleases are one of the most
important groups of enzymes for the
manipulation of DNA.
These are the bacterial enzymes that can
cut/split DNA (from any source) at specific sites.
They were first discovered in E.coli restricting
the replication of bacteriophages, by cutting
the viral DNA (The host E. coli DNA is
protected from cleavage by addition of
methyl groups).
Thus, the enzymes that restrict the viral
replication are known as restriction enzymes
or restriction endonucleases.
Recognition sequence is the site where the
DNA is cut by a restriction endonuclease.
Restriction endonucleases can specifically
recognize DNA with a particular sequence of
4-8 nucleotides & cleave.
Cleavage patterns: Majority of restriction
endonucleases (particularly type II) cut DNA
at defined sites within recognition sequence.
The cut DNA fragments by restriction
endonucleases may have mostly sticky ends
(cohesive ends) or blunt ends.
DNA fragments with sticky ends are useful for
recombinant DNA experiments.
This is because the single-stranded sticky DNA
ends can easily pair with any other DNA
fragment having complementary sticky ends.
The cut DNA fragments are covalently joined
together by DNA ligases.
These enzymes were originally isolated from
viruses.
They also occur in E.coli & eukaryotic cells.
DNA ligases actively participate in cellular
DNA repair process.
The hosts are the living systems or cells in
which the carrier of recombinant DNA
molecule or vector can be propagated.
There are different types of host cells-
prokaryotic (bacteria) & eukaryotic (fungi,
animals & plants).
Host cells, besides effectively incorporating
the vector's genetic material, must be
conveniently cultivated in the laboratory to
collect the products.
Microorganisms are preferred as host cells,
since they multiply faster compared to cells
of higher organisms (plants or animals).
Escherichia coli:
Escherichia coli was the first organism used
in the DNA technology & continues to be the
host of choice by many workers.
The major drawback is that E. coli (or even
other prokaryotic organisms) cannot
perform post-translational modifications.
Bacillus subtilis as an alternative to E.coli.
The most commonly used eukaryotic organism
is the yeast, Saccharomyces cerevisiae.
Certain complex proteins which cannot be
synthesized by bacteria can be produced by
mammalian cells e.g. tissue plasminogen
activator.
The mammalian cells possess the machinery to
modify the protein to the active form (post-
translational modifications).
Vectors are the DNA molecules, which can
carry a foreign DNA fragment to be cloned.
They are self-replicating in an appropriate
host cell.
The most important vectors are plasmids,
bacteriophages, cosmids & artificial
chromosome vectors.
Plasmids are extrachromosomal, double-
stranded, circular, self-replicating DNA
molecules.
Almost all bacteria have plasmids.
Size of plasmids varies from 1 to 500 kb.
Plasmids contribute to about 0.5 to 5.0% of
total DNA of bacteria.
pBR322 has a DNA sequence of 4,361 bp.
It carries genes resistance for ampicillin
(Amp1) & tetracycline (Tel1) that serve as
markers for the identification of clones
carrying plasmids.
The plasmid has unique recognition sites for
the action of restriction endonucleases - EcoRl,
Hindlll, BamHl, Sall & Pstll
The other plasmids employed as cloning
vectors include pUC19 (2,686 bp, with
ampicillin resistance gene) & derivatives of
pBR322-pBR325, pBR328 & pBR329.
Bacteriophages or phages are the viruses
that replicate within the bacteria.
In case of certain phages, their DNA gets
incorporated into the bacterial chromosome
& remains there permanently.
Phage vectors can accept short fragments of
foreign DNA into their genomes.
Phages can take up larger DNA segments
than plasmids.
Phage vectors are preferred for working
with genomes of human cells.
The most commonly used phages are
bacteriophage λ (phage λ) & bacteriophage
(phage M13).
Cosmids are victors possessing the characteristics of
both plasmid & phage λ.
Cosmids can be constructed by adding a fragment of
phage λ DNA including Cos site, to plasmids.
A foreign DNA (about 40 kb) can be inserted into
cosmid DNA .
The recombinant DNA, formed can be packed as
phages & injected into E.coli.
Inside host cell, cosmids behave like plasmids &
replicate & can carry larger fragments of foreign DNA
Human artificial chromosome (HAC):
Artificial chromosome is a synthetically
produced vector DNA, possessing the
characteristics of human chromosome.
HAC may be considered as a self-replicating
microchromosome with a size ranging from
1/10th to 1/5th of a human chromosome.
It can carry long human genes.
Yeast artificial chromosome (YAC) is a
synthetic DNA that can accept large
fragments of foreign DNA (particularly
human DNA).
It is possible to clone large DNA pieces by
using YAC.
Construction of BACs is based on one F-
plasmid which is larger than the other
plasmids used as cloning vectors.
BACs can accept DNA inserts of around 300 kb.
Transformation:
Transformation is the method of introducing
foreign DNA into bacterial cells (e.g. E.coli).
Uptake of plasmid DNA by E.coli is carried
out in ice-cold CaCl2 (0-5˚C) & a subsequent
heat shock (37-45˚C for about 90 sec).
A natural microbial recombination process.
During conjugation, two live bacteria (a
donor & a recipient) come together, join by
cytoplasmic bridges & transfer single
stranded DNA (from donor to recipient).
In side recipient cell, new DNA may integrate
with the chromosome or may remain free.
It is a technique involving electric field
mediated membrane permeabiIization.
Electric shocks can also induce cellular uptake
of exogenous DNA (believed to be via the
pores formed by electric pulses) from the
suspending solution.
It is a simple & rapid technique for
introducing genes into cells.
Liposomes are circular lipid molecules, which
have an aqueous interior that can carry
nucleic acids.
Several techniques have been developed to
encapsulate DNA in liposomes.
The liposome mediated gene transfer is
referred to as lipofection.
Treatment of DNA fragment with liposomes,
DNA pieces get encapsulated inside liposomes.
These liposomes can adhere to cell membranes
& fuse with them to transfer DNA fragments.
The DNA enters the cell & to the nucleus.
Positively charged liposomes efficiently
complex with DNA, bind to cells & transfer DNA
It is possible to directly transfer the DNA into
the cell nucleus.
Microinjection & particle bombardment are
the two techniques used for this purpose.
A clone refers to a group of organisms,
cells, molecules or other objects, arising
from a single individual.
Generation of DNA fragments
Insertion into a cloning vector
Introduction into host cells
Selection or screening
RE-digestion, cDNA synthesis, PCR, chemical synthesis
Ligation of blunt ends, homopolymertailing, linker molecules
Transformation, transfection, tradsduction
Hybridization, PCR, immunochemical methods, protein-protein interactions, functional complementation
Textbook of Biochemistry – U Satyanarayana
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