UNIT I –TOOLS AND TECHNIQUES OF GENETIC ENGINEERING

ENZYMES

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 restricting the replication of bacteriophages in

E.coli, 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.

Hundreds of restriction endonucleases have been isolated from bacteria, and some of them are

commercially available. The progress and growth of biotechnology is unimaginable without the

availability of restriction enzymes.

Nomenclature:

Restriction endonucleases are named by a standard procedure, with particular reference to the

bacteria from which they are isolated. The first letter (in italics) of the enzymes indicates the

genus name, followed by the first two letters (also in italics) of the species, then comes the

strain of the organism and finally a Roman numeral indicating the order of discovery. A couple

of examples are given below.

EcoRI is from Escherichia (E) coli (co), strain Ry13 (R) and first endonuclease (I) to be discovered.

Hindlll is from Haemophilus (H) influenza (in), strain Rd (d) and the third endonucleases (III) to

be discovered.

Types of endonucleases:

At least 4 different types of restriction endonucleases are known-type 1 (e.g Ecok12), type II

(e.g. EcoRI), type III (e.g. EcoPI) and type IIs. Their characteristic features are given in Table 6.1.

Among these, type II restriction endonucleases are most commonly used in gene cloning.

Recognition sequences:

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 and

cleave. Each recognition sequence has two fold rotational symmetry i.e. the same nucleotide

sequence occurs on both strands of DNA which run in opposite direction (Table 6.2). Such

sequences are referred to as palindromes, since they read similar in both directions (forwards

and backwards).

Cleavage patterns:

Majority of restriction endonucleases (particularly type II) cut DNA at defined sites within

recognition sequence. A selected list of enzymes, recognition sequences, and their products

formed is given in Table 6.2. The cut DNA fragments by restriction endonucleases may have

mostly sticky ends (cohesive ends) or blunt ends, as given in Table 6.2. DNA fragments with

sticky ends are particularly 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.

Reverse transcriptase

An enzyme, occurring in retroviruses, that catalyses the formation of double-stranded DNA

using the single RNA strand of the viral genome as template.

The enzyme is used in genetic engineering for producing complementary DNA from

messenger RNA.

DNA Ligases —DNA Joining Enzymes:

The cut DNA fragments are covalently joined together by DNA ligases. These enzymes were

originally isolated from viruses. They also occur in E.coli and eukaryotic cells. DNA ligases

actively participated in cellular DNA repair process.

The action of DNA ligases is absolutely required to permanently hold DNA pieces. This is so

since the hydrogen bonds formed between the complementary bases (of DNA strands) are not

strong enough to hold the strands together. DNA ligase joins (seals) the DNA fragments by

forming a phosphodiester bond between the phosphate group of 5′-carbon of one deoxyribose

with the hydroxyl group 3′-carbon of another deoxyribose (Fig. 6.2).

Phage T4 DNA ligase requires ATP as a cofactor while E.coli DNA ligase is dependent on NAD+. In

each case, the cofactor (ATP or NAD+) is split to form an enzyme—AMP complex that brings

about the formation of phosphodiester bond. The action of DNA ligase is the ultimate step in

the formation of a recombinant DNA molecule.

Alkaline Phosphatase:

Alkaline phosphatase is an enzyme involved in the removal of phosphate groups. This enzyme is

useful to prevent the unwanted ligation of DNA molecules which is a frequent problem

encountered in cloning experiments. When the linear vector plasmid DNA is treated with

alkaline phosphatase, the 5′-terminal phosphate is removed (Fig 6.5). This prevents both re-

circularization and plasmid DNA dimer formation. It is now possible to insert the foreign DNA

through the participation of DNA ligase.

Polymerases:

The groups of enzymes that catalyse the synthesis of nucleic acid molecules are collectively

referred to as polymerases. It is customary to use the name of the nucleic acid template on

which the polymerase acts (Fig. 6.6B). The three important polymerases are given below.

a. DNA-dependent DNA polymerase that copies DNA from DNA.

b. RNA-dependent DNA polymerase (reverse transcriptase) that synthesizes DNA from RNA.

c. DNA-dependent RNA polymerase that produces RNA from DNA

Klenow enzyme

Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains

polymerization and 3'→ 5' exonuclease activity, but has lost 5'→ 3' exonuclease activity.

Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.

CLONING VECTORS

Plasmids:

Plasmids are extra chromosomal, double- stranded, circular, self-replicating DNA molecules.

Almost all the bacteria have plasmids containing a low copy number (1-4 per cell) or a high copy

number (10-100 per cell). The size of the plasmids varies from 1 to 500 kb. Usually, plasmids

contribute to about 0.5 to 5.0% of the total DNA of bacteria (Note: A few bacteria contain linear

plasmids e.g. Streptomyces sp, Borella burgdorferi).

Types of plasmids:

There are many ways of grouping plasmids. They are categorized as conjugative if they carry a

set of transfer genes (tra genes) that facilitates bacterial conjugation, and non-conjugative, if

they do not possess such genes. Another classification is based on the copy number. Stringent

plasmids are present in a limited number (1-2 per cell) while relaxed plasmids occur in large

number in each cell.

F-plasmids possess genes for their own transfer from one cell to another, while R-plasmids

carry genes resistance to antibiotics. In general, the conjugative plasmids are large, show

stringent control of DNA replication, and are present in low numbers. On the other hand, non-

conjugative plasmids are small, show relaxed control of DNA replication, and are present in high

numbers.

Nomenclature of plasmids:

It is a common practice to designate plasmid by a lowercase p, followed by the first letter(s) of

researcher(s) names and the numerical number given by the workers. Thus, pBR322 is a

plasmid discovered by Bolivar and Rodriguez who designated it as 322. Some plasmids are given

names of the places where they are discovered e.g. pUC is plasmid from University of California.

pBR322 — the most common plasmid vector:

pBR322 of E.coli is the most popular and widely used plasmid vector, and is appropriately

regarded as the parent or grandparent of several other vectors. pBR322 has a DNA sequence of

4,361 bp. It carries genes resistance for ampicillin (Ampr) and tetracycline (TeIr) that serve as

markers for the identification of clones carrying plasmids. The plasmid has unique recognition

sites for the action of restriction endonucleases such as EcoRI, Hindlll, BamHI, Sail and Pstll (Fig.

6.8).

Other plasmid cloning vectors:

The other plasmids employed as cloning vectors include pUC19 (2,686 bp, with ampicillin

resistance gene), and derivatives of pBR322- pBR325, pBR328 and pBR329.

Bacteriophages:

Bacteriophages or simply phages are the viruses that replicate within the bacteria. In case of

certain phages, their DNA gets incorporated into the bacterial chromosome and remains there

permanently. Phage vectors can accept short fragments of foreign DNA into their genomes. The

advantage with phages is that they can take up larger DNA segments than plasmids. Hence

phage vectors are preferred for working with genomes of human cells.

Bacteriophage λ:

Bacteriophage lambda (or simply phage λ), a virus of E.coli, has been most thoroughly studied

and developed as a vector. In order to understand how bacteriophage functions as a vector, it is

desirable to know its structure and life cycle (Fig. 6.9).

Phage λ consists of a head and a tail (both being proteins) and its shape is comparable to a

miniature hypodermic syringe. The DNA, located in the head, is a linear molecule of about 50

kb. At each end of the DNA, there are single-stranded extensions of 12 base length each, which

have cohesive (cos) ends.

On attachment with tail to E.coli, phage X injects its DNA into the cell. Inside E.coli, the phage

linear DNA cyclizes and gets ligated through cos ends to form a circular DNA. The phage DNA

has two fates-lytic cycle and lysogenic cycle.

Lytic cycle:

The circular DNA replicates and it also directs the synthesis of many proteins necessary for the

head, tail etc., of the phage. The circular DNA is then cleaved (to form cos ends) and packed

into the head of the phage. About 100 phage particles are produced within 20 minutes after the

entry of phage into E.coli.

The host cell is then subjected to lysis and the phages are released. Each progeny phage particle

can infect a bacterial cell, and produce several hundreds of phages. It is estimated that by

repeating the lytic cycle four times, a single phage can cause the death of more than one billion

bacterial cells.

If a foreign DNA is spliced into phage DNA, without causing harm to phage genes, the phage will

reproduce (replicate the foreign DNA) when it infects bacterial cell. This has been exploited in

phage vector employed cloning techniques.

Lysogenic cycle:

In this case, the phage DNA (instead of independently replicating) becomes integrated into the

E.coli chromosome and replicates along with the host genome. No phage particles are

synthesized in this pathway.

Use of phage λ as a vector:

Only about 50% of phage λ DNA is necessary for its multiplication and other functions. Thus, as

much as 50% (i.e. up to 25kb) of the phage DNA can be replaced by a donor DNA for use in

cloning experiments. However, several restriction sites are present on phage λ which is not by

itself a suitable vector. The λ-based phage vectors are modifications of the natural phage with

much reduced number of restriction sites.

Insertion vectors:

They have just one unique cleavage site, which can be cleaved, and a foreign DNA ligated in. It

is essential that sufficient DNA (about 25%) has to be deleted from the vector to make space for

the foreign DNA (about 18kb).

Replacement vectors:

These vectors have a pair of restriction sites to remove the non-essential DNA (stuffer DNA)

that will be replaced by a foreign DNA. Replacement vectors can accommodate up to 24kb, and

propagate them. Many phage vector derivations (insertion/ replacement) have been produced

by researchers for use in recombinant DNA technology.

The main advantage of using phage vectors is that the foreign DNA can be packed into the

phage {in vitro packaging), the latter in turn can be injected into the host cell very effectively

(Note: No transformation is required).

Cosmids:

Cosmids are the vectors possessing the characteristics of both plasmid and bacteriophage 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 so formed can be packed as phages and injected into E.coli (Fig. 6.10).

Once inside the host cell, cosmids behave just like plasmids and replicate. The advantage with

cosmids is that they can carry larger fragments of foreign DNA compared to plasmids.

Artificial Chromosome Vectors:

Human artificial chromosome (HAC):

Developed in 1997 (by H. Willard), human artificial chromosome is a synthetically produced

vector DNA, possessing the characteristics of human chromosome. HAC may be considered as a

self-replicating micro-chromosome with a size ranging from 1/10th to 1/5th of a human

chromosome. The advantage with HAC is that it can carry human genes that are too long.

Further, HAC can carry genes to be introduced into the cells in gene therapy.

Bacterial artificial chromosomes (BACs):

The 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. The advantage with

bacterial artificial chromosome is that the instability problems of YACs can be avoided. In fact, a

major part of the sequencing of human genome has been accomplished by using a library of

BAC recombinant.

Yeast artificial chromosomes (YACs):

Introduced in 1987 (by M. Olson), yeast artificial chromosome (YAC) is a synthetic DNA that can

accept large fragments of foreign DNA (particularly human DNA). It is thus possible to clone

large DNA pieces by using YAC. YACs are the most sophisticated yeast vectors, and represent

the largest capacity vectors available. They possess centromeric and telomeric regions, and

therefore the recombinant DNA can be maintained like a yeast chromosome.

Shuttle Vectors:

The plasmid vectors that are specifically designed to replicate in two different hosts (say in

E.coli and Streptomyces sp) are referred to as shuttle vectors. The origins of replication for two

hosts are combined in one plasmid. Therefore, any foreign DNA fragment introduced into the

vector can be expressed in either host. Further, shuttle vectors can be grown in one host and

then shifted to another host (hence the name shuttle). A good number of eukaryotic vectors

are shuttle vectors.

HOST CELLS – ADVANTAGES AND LIMITATIONS

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) and

eukaryotic (fungi, animals and plants). Some examples of host cells used in genetic engineering

are given in Table 6.4.

Host cells, besides effectively incorporating the vector’s genetic material, must be conveniently

cultivated in the laboratory to collect the products. In general, microorganisms are preferred as

host cells, since they multiply faster compared to cells of higher organism (plants or animals).

Prokaryotic Hosts:

Escherichia coli:

The bacterium, Escherichia coli was the first organism used in the DNA technology experiments

and continues to be the host of choice by many workers. Undoubtedly, E.coli, the simplest

Gram negative bacterium (a common bacterium of human and animal intestine), has played a

key role in the development of present day biotechnology.

Under suitable environment, E.coli can double in number every 20 minutes. Thus, as the

bacteria multiply, their plasmids (along with foreign DNA) also multiply to produce millions of

copies, referred to as colony or in short clone. The term clone is broadly used to a mass of cells,

organisms or genes that are produced by multiplication of a single cell, organism or gene.

Limitations of E. coli:

There are certain limitations in using E.coli as a host. These include- causation of diarrhea by

some strains, formation of endotoxins that are toxic, and a low export ability of proteins from

the cell. Another major drawback is that E.coli (or even other prokaryotic organisms) cannot

perform post-translational modifications.

Bacillus subtilis:

Bacillus subtilis is a rod shaped non-pathogenic bacterium. It has been used as a host in industry

for the production of enzymes, antibiotics, insecticides etc. Some workers consider B.subtilis as

an alternative to E.coli.

Eukaryotic Hosts:

Eukaryotic organisms are preferred to produce human proteins since these hosts with complex

structure (with distinct organelles) are more suitable to synthesize complex proteins. The most

commonly used eukaryotic organism is the yeast, Saccharomyces cerevisiae. It is a non-

pathogenic organism routinely used in brewing and baking industry. Certain fungi have also

been used in gene cloning experiments.

Mammalian cells:

Despite the practical difficulties to work with and high cost factor, mammalian cells (such as

mouse cells) are also employed as hosts. The advantage is that certain complex proteins which

cannot be synthesized by bacteria can be produced by mammalian cells e.g. tissue plasminogen

activator. This is mainly because the mammalian cells possess the machinery to modify the

protein to its final form (post-translational modifications).

It may be noted here that the gene manipulation experiments in higher animals and plants are

usually carried out to alter the genetic makeup of the organism to create transgenic animals

and transgenic plants rather than to isolate genes for producing specific proteins.

ISOLATION AND PURIFICATION OF NUCLEIC ACIDS (GENOMIC/PLASMID DNA

AND RNA)

Every gene manipulation procedure requires genetic material like DNA and RNA. Nucleic acids

occur naturally in association with proteins and lipoprotein organelles. The dissociation of a

nucleoprotein into nucleic acid and protein moieties and their subsequent separation, are the

essential steps in the isolation of all species of nucleic acids.

Isolation of nucleic acids is followed by quantitation of nucleic acids generally done by either

spectrophotometric or by using fluorescent dyes to determine the average concentrations and

purity of DNA or RNA present in a mixture.

Isolating the genetic material (DNA) from cells (bacterial, viral, plant or animal) involves three

basic steps-

• Rupturing of cell membrane to release the cellular components and DNA

• Separation of the nucleic acids from other cellular components

• Purification of nucleic acids

Isolation and Purification of Genomic DNA

Genomic DNA is found in the nucleus of all living cells with the structure of double stranded

DNA remaining unchanged (helical ribbon). The isolation of genomic DNA differs in animals and

plant cells. DNA isolation from plant cells is difficult due to the presence of cell wall, as

compared to animal cells. The amount and purity of extracted DNA depends on the nature of

the cell.

The method of isolation of genomic DNA from a bacterium comprises following steps –

1. Bacterial culture growth and harvest.

2. Cell wall rupture and cell extract preparation.

3. DNA Purification from the cell extract.

4. Concentration of DNA solution.

Growth and harvest of bacterial culture

Bacterial cell culture is more convenient than any other microbe, as it requires only liquid

medium (broth) containing essential nutrients at optimal concentrations, for the growth and

division of bacterial cells. The bacterial cells are usually grown on a complex medium like Luria-

Bertani (LB), in which the medium composition is difficult to decipher. Later, the cells are

separated by centrifugation and resuspended in 1% or less of the initial culture volume.

Preparation of cell extract

Bacterial cell is surrounded by an additional layer called cell wall, apart from plasma membrane

with some species of E. coli comprising multilayered cell wall. The lysis of cell wall to release the

genetic material i.e. DNA can be achieved by following ways-

• Physical method by mechanical forces.

• Chemical method by metal chelating agents i.e. EDTA and surfactant i.e. SDS or enzyme (e.g.

lysozyme). Lysozyme • present in egg-white, salivary secretion and tears. • catalyzes the

breakdown of cell wall i.e. the peptidoglycan layer. EDTA (Ethylene diamine tetra-acetic acid) •

a chelating agent necessary for destabilizing the integrity of cell wall. • inhibits the cellular

enzymes that degrade DNA. SDS (Sodium dodecyl sulphate) • helps in removal of lipid

molecules and denaturation of membrane proteins. Generally, a mixture of EDTA and lysozyme

is used. Cell lysis is followed by centrifugation to pellet down the cell wall fractions leaving a

clear supernatant containing cell extract.

Purification of DNA

In addition to DNA, a cell extract contains significant quantities of protein and RNA which can

be further purified by following methods.

Organic extraction and enzymatic digestion for the removal of contaminants

It involves the addition of a mixture of phenol and chloroform (1:1) to the cell lysate for protein

separation. The proteins aggregate as a white mass in between the aqueous phase containing

DNA and RNA, and the organic layer. Treatment of lysate with pronase or protease, in addition

to phenol/chloroform, ensures complete removal of proteins from the extract. The RNA can be

effectively removed by using Ribonuclease, an enzyme which rapidly degrades RNA into its

ribonucleotide subunits. Repeated phenol extraction is not desirable, as it damages the DNA. 4-

1.2.3.2. Using ion-exchange chromatography This involves the separation of ions and polar

molecules (proteins, small nucleotides and amino acids) based on their charge. DNA carrying

negative charge binds to the cationic resin or matrix which can be eluted from the column by

salt gradient. Gradual increase in salt concentration detaches molecules from the resin one

after another. Figure 4-1.2. Preparation of genomic DNA

Concentration of DNA samples

Concentration of DNA can be done using ethanol along with salts such as sodium acetate,

potassium acetate etc. These salts provide metal ions like sodium ions (Na+), potassium ions

(K+) which help in aggregation and hence precipitation of DNA molecules. Advantage It leaves

short-chain and monomeric nucleic acid components in solution. Ribonucleotides produced by

the ribonuclease treatment are separated from DNA. 4-1.3. Isolation and Purification of Plasmid

DNA Plasmids are circular, double stranded extra cellular DNA molecules of bacterium and most

commonly used in recombinant DNA technology. The isolation of plasmid DNA involves three

major steps1. Growth of the bacterial cell. 2. Harvesting and lysis of the bacteria. 3. Purification

of the plasmid DNA. 4-1.3.1. Growth of the bacterial cell It involves growth of the bacterial cells

in a media containing essential nutrients. 4-1.3.2. Harvest and lysis of bacteria Lysis of bacteria

results in the precipitation of DNA and cellular proteins. Addition of acetate-containing

neutralization buffer results in the precipitation of large and less supercoiled chromosomal DNA

and proteins leaving the small bacterial DNA plasmids in solution. 4-1.3.3. Purification of

Plasmid DNA This step is same for both plasmid and genomic but former involves an additional

step i.e. the separation of plasmid DNA from the large bacterial chromosomal DNA..

Methods for separation of plasmid DNA

Separation of plasmid DNA is based on the several features like size and conformation of

plasmid DNA and bacterial DNA. Plasmids are much smaller than the bacterial main

chromosomes, the largest plasmids being only 8% of the size of the E. coli chromosome. The

separation of small molecules (i.e. plasmids) from larger ones (i.e. bacterial chromosome) is

based on the fact that plasmids and the bacterial chromosomes are circular but bacterial

chromosomes break into linear fragments during the preparation of the cell extract resulting in

separation of pure plasmids. The methods of separation of plasmid DNA are described as

below4-1.3.3.1.1. Separation based on size difference • It involves lysis of cells with lysozyme

and EDTA (function as described above in point 4-1.2.2.) in the presence of sucrose (prevents

the immediate bursting of cell). • Cells with partially degraded cell walls are formed that retain

an intact cytoplasmic membrane called as sphaeroplasts. • Cell lysis is then induced by the

addition of a non-ionic detergent (e.g. Triton X100) or ionic detergents (e.g. SDS) causing

chromosomal breakage. • Bacterial chromosome attached to cell membrane, upon lysis gets

removed with the cell debris. • A cleared lysate consisting almost entirely of plasmid DNA is

formed with very little breakage of the bacterial DNA. (Figure 4-1.3.3.1.1.).

Separation of plasmid DNA on the basis of size.

Separation based on conformation

Plasmids are supercoiled molecules formed by partial unwinding of double helix of the plasmid

DNA during the plasmid replication process by enzymes called topoisomerases. The supercoiled

conformation can be maintained when both polynucleotide strands are intact, hence called

covalently closed-circular (ccc) DNA. If one of the polynucleotide strands is broken, the double

helix reverts to its normal relaxed state taking an alternative conformation, called open-circular

(oc). Super coiling is important in plasmid preparation due to the easy separation of supercoiled

molecules from non-supercoiled ones.

The commonly used methods of separation based on conformation are as follows4-1.3.3.1.2

(a). Alkaline denaturation method

• This method is based on maintaining a very narrow pH range for the denaturation of non-

supercoiled DNA but not the supercoiled plasmid (Figure 4-1.3.3.1.2(a).). • Addition of sodium

hydroxide to cell extract or cleared lysate (pH12.0-12.5) results in disruption of the hydrogen

bonds of non-supercoiled DNA molecules. • As a result, the double helix unwinds and two

polynucleotide chains separate. • Further addition of acid causes the aggregation of these

denatured bacterial DNA strands into a tangled mass which can be pelleted by centrifugation,

leaving plasmid DNA in the supernatant. Advantage • Most of the RNA and protein under

defined conditions (specifically cell lysis by SDS and neutralization with sodium acetate) can be

removed by the centrifugation. • No requirement of organic extraction.

Separation of plasmid DNA by Alkaline denaturation method 4-1.3.3.1.2(b). Ethidium bromide-

cesium chloride density gradient centrifugation • Density gradient centrifugation can separate

DNA, RNA and protein. It is a very efficient method for obtaining pure plasmid DNA. • A density

gradient is produced by centrifuging a solution of cesium chloride at a very high speed which

pulls the CsCl ions towards the bottom. This process is referred as isopycnic centrifugation. •

The DNA migrates to the point at which it has density similar to that of CsCl i.e.1.7 g/cm3 in the

gradient. • In contrast, protein molecules having lower buoyant densities float at the top of the

tube whereas RNA gets pelleted at the bottom. Density gradient centrifugation in the presence

of ethidium bromide (EtBr) can be used to separate supercoiled DNA from non-super coiled

molecules. Ethidium bromide is an intercalating dye that binds to DNA molecules causing partial

unwinding of the double helix. Supercoiled DNA have very little freedom to unwind due to

absence of free ends and bind to a limited amount of EtBr resulting in very less decrease in

buoyant density (0.085 g/cm3 ) than that of linear DNA (0.125 g/cm3 ). As a result, they form a

distinct band separated from the linear bacterial DNA. The EtBr bound to DNA is then extracted

by n-butanol and the CsCl is removed by dialysis. 4-1.4.

Isolation and Purification of RNA

RNA (Ribonucleic acid) is a polymeric substance consisting of a long single-stranded chain of

phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine and uracil

bonded to the ribose sugar present in living cells and many viruses. The steps for preparation of

RNA involve homogenization, phase separation, RNA precipitation, washing and re-dissolving

RNA. The method for isolation and purification of RNA are as follows

1) Organic extraction method

2) Filter-based, spin basket formats

3) Magnetic particle methods

4) Direct lysis method.

Organic extraction method This method involves phase separation by addition and

centrifugation of a mixture of a solution containing phenol, chloroform and a chaotropic agent

(guanidinium thiocyanate) and aqueous sample. Guanidium thiocyanate results in the

denaturation of proteins and RNases, separating rRNA from ribosomes. Addition of chloroform

forms a colorless upper aqueous phase containing RNA, an interphase containing DNA and a

lower phenol-chloroform phase containing protein. RNA is collected from the upper aqueous

phase by alcohol (2-propanol or ethanol) precipitation followed by rehydration.

METHODS OF GENE TRANSFER:

The six methods are: (1) Transformation (2) Electroporation (3) Liposome-Mediated Gene

Transfer (4) Transduction and (5) Direct Transfer of DNA.6) Particle bombardment

1. Transformation:

Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli). The

uptake of plasmid DNA by E.coli is carried out in ice-cold CaCl2 (0-5°C), and a subsequent heat

shock (37-45°C for about 90 sec). By this technique, the transformation frequency, which refers

to the fraction of cell population that can be transferred, is reasonably good e.g. approximately

one cell for 1000 (10-3) cells.

Transformation efficiency:

It refers to the number of trans-formants per microgram of added DNA. For E.coli,

transformation by plasmid, the transformation efficiency is about 107 to 108 cells per

microgram of intact plasmid DNA. The bacterial cells that can take up DNA are considered as

competent. The competence can be enhanced by altering growth conditions.

The mechanism of the transformation process is not fully understood. It is believed that the

CaCI2 affects the cell wall, breaks at localized regions, and is also responsible for binding of DNA

to cell surface. A brief heat shock (i.e. the sudden increase in temperature from 5°C to 40°C)

stimulates DNA uptake. In general, large-sized DNAs are less efficient in transforming.

Other chemical methods for transformation:

Calcium phosphate (in place of CaCI2) is preferred for the transfer of DNA into cultured cells.

Sometimes, calcium phosphate may result in precipitate and toxicity to the cells. Some workers

use diethyl amino ethyl dextran (DEAE -dextran) for DNA transfer.

2. Electroporation:

Electroporation is based on the principle that high voltage electric pulses can induce cell plasma

membranes to fuse. Thus, electroporation is a technique involving electric field-mediated

membrane permeabilization. Electric shocks can also induce cellular uptake of exogenous DNA

(believed to be via the pores formed by electric pulses) from the suspending solution.

Electroporation is a simple and rapid technique for introducing genes into the cells from various

organisms (microorganisms, plants and animals).

The basic technique of electroporation for transferring genes into mammalian cells is depicted

in Fig. 6.11. The cells are placed in a solution containing DNA and subjected to electrical shocks

to cause holes in the membranes. The foreign DNA fragments enter through the holes into the

cytoplasm and then to nucleus.

Electroporation is an effective way to transform E.coli cells containing plasmids with insert

DNAs longer than 100 kb. The transformation efficiency is around 109 transformants per

microgram of DNA for small plasmids (about 3kb) and about 106 for large plasmids (about 130

kb).

3. Liposome-Mediated Gene Transfer:

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, referred to as lipofection, is depicted in Fig. 6.12.

On treatment of DNA fragment with liposomes, the DNA pieces get encapsulated inside

liposomes. These liposomes can adher to cell membranes and fuse with them to transfer DNA

fragments. Thus, the DNA enters the cell and then to the nucleus. The positively charged

liposomes very efficiently complex with DNA, bind to cells and transfer DNA rapidly.

Lipofection is a very efficient technique and is used for the transfer of genes to bacterial, animal

and plant cells. T

4. Transduction:

Sometimes, the foreign DNA can be packed inside animal viruses. These viruses can naturally

infect the cells and introduce the DNA into host cells. The transfer of DNA by this approach is

referred to as transduction.

5. Direct Transfer of DNA:

It is possible to directly transfer the DNA into the cell nucleus. Microinjection and particle

bombardment are the two techniques commonly used for this purpose.

Microinjection:

DNA transfer by microinjection is generally used for the cultured cells. This technique is also

useful to introduce DNA into large cells such as oocytes, eggs and the cells of early embryos.

The term transfection is used for the transfer DNA into eukaryotic cells, by various physical or

chemical means.

6.Transfection by particle bombardment

Particle bombardment (also known as biolistics or microprojectile transfection) procedure

involves coating micrometer-sized gold or tungsten particles with DNA and then accelerating

the particles into cells or tissues. A major advantage of this method is that DNA can be

delivered to deep cells in tissue slices, and the depth of penetration can be adjusted by

changing the applied force. The size and total mass of the particles and the force of the

bombardment are important parameters that balance efficient penetration against cell

damage. The technique was developed for the transformation of maize and is now a method of

choice for generating transgenic cereal plants. For animal cells, the technique has been less

widely used because it is usually simpler to transfect cultured cells by alternative well-

established methods. However, the technique has found a role in the transfection of whole

organs and tissue slices, and more recently for the transfer of DNA to surface organs in gene

therapy.