 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, homopolymer
tailing, linker molecules
Transformation, transfection,
tradsduction
Hybridization, PCR, immunochemical methods, protein-
protein interactions, functional complementation

 Textbook of Biochemistry – U Satyanarayana