- After the cutting of DNA by restriction enzymes, fragments of DNA are formed.
- Separation of DNA fragments according to their size or length is done by a technique called gel electrophoresis developed by A. Tiselius in 1937.
- Electrophoresis is a technique of separation of molecules such as DNA RNA or protein on the basis of their size, under the influence of an electrical field, so that they migrate in the direction of electrode bearing the opposite charge, viz . , positively charged molecules move towards cathode (-ve electrode) and negatively charged mol ecules travel towards anode (+ ve elec trode) through a medium/ matrix.
- Electrophoresis performed in a gel matrix, so that molecules of similar electric charge can be separated on the basis of size, is called gel electrophoresis.
- Now a days the most commonly used matrix is agarose which is a polysac charide extracted from sea weeds.
- DNA fragments separate according to size through the pores of agarose gel.
- Hence the smaller the fragment size the farther it moves.
- Agarose dissolves in hot water when this solution is cooled, double helices form and become arranged laterally and produce thick filaments.
- These filaments become cross linked to form the gel.
- Pore size depends on agarose concentration.
- The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (EtBr ) followed by exposure to UV radiation as bright orange coloured bands.
- The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.
- This process is called as elution (removal of adsorbent).
- These purified DNA fragments are used in constructing recombinant DNA by linking them with cloning vectors.
Cloning Vectors (Vehicle DNA or carrier of DNA)
- The vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell.
- Vectors may be plasmids, a bacteriophages (viruses that attack bacteria), cosmids, Yeast artificial chromosomes (YACs), Bacterial artificial chromosomes (BACs) and viruses.
- There are also some shuttle vectors.
- Out of these vectors, the most commonly used cloning vectors are plasmids and viruses.
- Plasmid Vectors
- Plasmids were discovered by Willium Hays and Joshua Lederberg (1952).
- These are extra - chromosomal, self - replicating, usually circular, double - stranded DNA molecules, found naturally in many bacteria and also in some yeast.
- Although plasmids are usually not essential for normal cell growth and division, they often confer some traits on the host organism, for example, resistance to certain antibiotics or toxins that can be selective advantage under certain conditions.
- The plasmid molecules may be present as 1 or 2 copies or in multiple copies (500-700) inside the host organism.
- These naturally occurring plasmids have been modified to serve as vectors in the laboratory.
- The most widely used, versatile, easily manipu lated vector PBR 322 is an ideal plasmid vector.
- PBR322 Vector
- This was the first artificial cloning vector constructed in 1977 by Boliver and Rodriguez.
- It is widely used in gene cloning experiments.
- Nomenclature : In PBR 322 plasmid
- P - denotes that it is a plasmid
- BR - stands for Boliver and Rodriguez who constructed this plasmid.
- 322 is a number given to distinguish this plasmid from others developed in the same laboratory.
- For example, there are plasmids PBR 325, PBR 327, PBR 328 PBR 345 etc.
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