Following the fragmentation of the DNA, the fragments are to be separated by a technique known as electrophoresis.

Electrophoresis: It is a technique of separating electrically charged molecules of different molecular weights in an electric field. DNA, RNA and protein molecules of differing molecular sizes are separated from their respective mixtures by this technique.

There are two types of electrophoresis techniques: paper and gel. The one that is used in molecular biology is the gel electrophoresis. Here, a jelly like porous and semisolid medium is the medium of migration of the molecules in an electric field. Therefore, the gel is often called molecular sieve. Two types of gels are used for molecular separations: agarose and polyacrylamide. Agarose is used for molecular separation of nucleic acids (RNA and DNA), while polyacrylamide for proteins.

The rate of migration is inversely proportional to the molecular weight of the separating molecules and directly proportional to the strength of the electric current. Larger molecules migrate relatively slower, while smaller molecules migrate faster.

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The migration is towards the anode (positively charged electrode). Therefore, the migrating molecules are negatively charged. Nucleic acids can be conferred uniform negative charges in a buffer solution. This type of electrophoresis is known as agarose gel electrophoresis. The separated DNA fragments are not visible.

These have to be stained with a dye for resolving the distance of migration. From the distance a fragment has traveled, its molecular weight (size) can be determined by comparing with that of a standard. This stain is known as ethidium bromide (EtBr).

It is an intercalating agent i.e. it inserts between two consecutive base pairs. EtBr absorbs UV light at a wavelength of 260 nm and fluoresces orange. Another dye, bromophenol blue is used as a marker dye to monitor the extent of migration of the DNA fragments during electrophoresis.

However, a protein molecule contains many amino acids and all the amino acids do not have similar acid-base properties. Hence, a protein molecule cannot be conferred with uniform negative charges in a buffer solution.

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It is therefore, treated with SDS, an anionic detergent that coats a protein molecule and confers negative charges uniformly. The protein electrophoresis is known as Sodium codicil sulphate – polyacrylamide gel electrophoresis (SDS – PAGE).

The arrow indicates the direction of migration of DNA fragments, (be) Fluorescent bands on an agarose gel slab containing DNA fragments differing molecular weights. (Note that the migration takes place from cathode (-) to anode and the rate of migration is inversely proportional to the molecular weight of the fragments).

1. Prokaryotic Hosts and Vectors:

(a) Host: A gene does not have an intrinsic ability to replicate. Therefore, its joined to another DNA segment having a spontaneous property of replica; this segment of DNA is known as a vector DNA.

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The resultant Dynamo by the joining of the gene and the vector DNA is the recombinant/chi DNA. It will replicate only in a suitable environment, where all fondant infrastructural facilities are available. These facilities are provided by a host the most suitable host cell is E. coli K12 (K12 being the strain).

(be) Vector: A vector is a DNA molecule, which carries a gene (DNA) for clod in a host cell. It is also known as a cloning vehicle. A suitable vector h following properties: (1) It is a DNA molecule. (2) It has an origin of replica (3) It has one or more genetic markers. (4) It has a cloning site at which the of interest will be inserted.

Several types of vectors are used in the cloning of a gene in a prokaryotic cell be the size of the insert: (i) Plasmids, (ii) Bacterial artificial chromosomes (BAC) (iii) and RNA bacteriophages and (iv) Hybrid plasmid – phage vector (Cosmos). You of the vector is very important in gene cloning.

(i) Plasmid: Plasmids are small 1 and double stranded extra chromosomal DNA mole having an origin of replication. They occur naturally in bacteria. Naturally occur plasmids are not suitable for cloning of genes.

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They are genetically engineered for making them suitable for cloning. Such plasmids are called cloning plasmids. A suitable cloning plasmid should preferably have the following properties: (i) it should be non-conjugative; (ii) it should have a relaxed replication and a high copy number

Cloning Plasmid: The commonest plasmid used for cloning genes is designated as purr 322

where up stands for the word plasmid and B and R signify the names of its discoverer

322 is a numeral designation that has a relevance to these workers, who worked out the plasmid.

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Features of pBR322: pBR322 has the following features:

1. It is a small double stranded circular DNA molecule.

2. It has an origin of replication, ore.

3. It has two antibiotic resistance marker genes: (i) ampicillin resistance gene (amp) and (ii) tetracycline resistance gene (tee).

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4. A unique Eco RI restriction site has been worked out into the segment between the two antibiotic resistance genes.

5. A unique-site for each of PST I and Put I is engineered into the amp.

6. One each of the restriction sites of Hind III, and Sal I is engineered into the tee gene.

(M) Bacteria] Artificial Chromosome (BAC): Plasmids have proved to be very good vectors for inserts of up to 10 kb. Some inserts of eukaryotic origin are much larger and show structural instability in plasmids. A variety of vectors are flow available for the cloning of eukaryotic genes.

Some of these are discussed in this and the followings’ Bacterial artificial chromosome (BAC) is one among these vectors. It is essential! bacterial plasmid to which a large segment of a human DNA is annealed. Thee number of this vector is kept at a low to overcome the problem of instability.

The clear insertion and ligation processes are similar to that of a bacterial plasmid. An exam a BAC is provided by an I (fertility) plasmid onto which is attached a large sigma a human DNA. An insert (>300 kb) may be inserted into a restriction site in as’ manner.

(iii) Bacteriophage Vector: Bacteriophages or simply phages are viruses, which in. bacteria and replicate their DNA inside the bacterial host. Sometimes, the phage gen integrates into the host genome and replicates once with every cycle of the host gen I replication. This phase of the life cycle is known as lysogeny. The integrated ph genome is known as a prophage. On induction, the phage genome detaches from of the host and undergoes several cycles of replication and forms multiple copies.” copy of the phage genome assembles into a fully formed phage particle and when a 1 number of such phage particles are formed, the host cell ruptures giving way to assembled phage particles. This part of the life cycle is known as lysis. The genome several phages is used as vectors in gene cloning.

The genome of the 1 phage has been proved to be a very good vector. Its genomei: double stranded linear DNA, 48.5 kb in length with a 12 bp overhanging single strand at eac end. The two single stranded ends are mutually cohesive i.e. they can undergo complement base pairing by hydrogen bonding.

The linear DNA gets jnto the host cell and circular^ spontaneously by the joining of the two cohesive ends. The site formed at the two cohesivee is known as the cos (cohesive) site. It then integrates into the host genome at another site recombination. When induced, it is detached from the host genome and undergoes replication rolling circle model and forms aconcatemer (several replicating DNA molecules joined h to tail).

Then a viral particle is formed with the assembly of exactly one segment of theDfi (from one cos site to another) with the preassembled protein coat and tail.

A large segment in the central region of the 1 genome, approximately one third the len^ of the entire genome, has genes that are not connected with productive infection or lysis, region is known as the central dispensable /stuffer region.

This region can be replacedb foreign DNA of a proportionate length so that the length of the recombinant DNA is betw 75% -105% of the length of the normal genome. Then the recombinant DNA id packaged vitro into a viral particle. E. coli cells are infected by this viral particle. A successful infect’

A single stranded DNA virus, Ml 3 is also used as a vector. The replicative form of genome is doubling stranded and circular. This form is used as a vector, as it is moreeas manipulated like that of a plasmid.

(iv) Plasmid – Phage hybrid:

A cosmid is ajoint venture of aclotr plasmid and the cos site of a 1 phage genome. The plasmid part has an origiaof replica genetic markers and a few unique restriction sites. A cos site of a 1 phage Di concatemer is removed by an appropriate RE digestion and annealed to the plasmid results in a circular cosmid vector. The cosmid is cleaved by an appropriate RE and insert is inserted and the circle is sealed.

Then it is linearized by opening it at the cos/ Several linearized cosmids are joined together to form a concatemer. The cosmids packaged into 1 prticles in vitro. Then E. coli are infected by such recombinan particles. Following infection, several daughter 1 particles are formed and releas the lysis of the host. Each 1 particle has a copy of the recombinant DNA and henc copy of the gene insert. Cosmids can accept DNA inserts in a range of 30 – 45 kb.

2. Eukaryotic Hosts and Vectors:

Plasmids can hold a small segment of (upto 10 kb) DNA for cloning. However, mo the eukaryotic genes are in the range of 200 – 2000 kb in length. Bacterial plas cannot hold these genes in a stable manner. Therefore, the host-vector requirement changes for cloning eukaryotic genes. Yeast (Saccharomyces cerevisiae) is the most commonly chosen host cell and yeast artificial chromosome (YAC) as the vector.

Eukaryotic genes may not function properly in a prokaryotic cell as they do in their normal environment. Therefore, it is difficult to clone eukaryotic genes in a prokaryotic host cell. Secondly, in the event of a successful cloning of a gene, it may not be expressed as a fully functional polypeptide product in a prokaryotic host cell.

Such genes can be cloned and expressed efficiently in eukaryotic host cells. Saccharomyces cerevisiae is the most favoured eukaryotic host cell for eukaryotic gene cloning. Plant and animal cells grown in cultures are also used as host cells.

Yeast Artificial Chromosome:

A YAC (8 kb) can hold a very long stretch of DNA (200 kb – 2000 kb). Unlike other vector systems, the stability of YAC increases with an increase in the size of the insert. It is genetically engineered by joining several segments together:

1. Autonomous replicating sequence (ARS) element of yeast for initiation and elongation of replication of the artificial chromosome.

2. Sequences from around the centromere (CEN sequence) of yeast for equal segregation to daughter cells following cell division.

3. Telomeres at the ends of the artificial chromosome for complete replication of the linear artificial chromosome and protection of the ends from nuclease digestion.

PYAC2 is a typical YAC vector. It is essentially a pBR322 into which a number of yeast genes such as TRP1, URA3 and CEN4 have been inserted. TRP1 carries an origin of replication. CEN4 sequence is from around the centromere of fourth chromosome of yeast.

The telomeres are provided by two sequences, TEL. Another sequence, SUP4 is worked out between CEN4 and URA3. This sequence acts as a selectable marker. Three restriction sites, two Bam HI and a Smal are engineered into the plasmid. The foreign gene is inserted into the SUP4 at Smal site.

3. Shuttle Vector:

A shuttle vector is compatible in both the prokaryotic and eukaryotic hosts. For compatibility, a shuttle vector must have two origins of replication, and two selectable markers, one for each host. The yeast episomal plasmid (YEp), pJDB219 is an example of a shuttle vector that can shuttle between E. coli and yeast.

4. Eukaryotic Hosts and Vectors (Animals):

Animal cells grown in culture are often the targets of transformation by exogenous DNA. Many animal viruses are used as vectors in the transformation of animal cells. A few commonly used animal viral vectors are simian virus 40 (SV 40), papilloma virus, baculovirus and retroviruses.