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