[Ishani7]

Molecular Biological methods are dealing with isolation, amplification, modification, identification of genetic material. These procedures make use of biological tools such as enzymes, primers, plasmids etc. Most of these are isolated from microbes which are of great importance. Ability of these enzymes and other compounds to withstand different in-vitro conditions is the reason why they are used for experimental purpose.

DNA polymerase is the master blaster enzyme used in molecular methods such as Polymerase Chain Reaction (PCR). This enzyme catalyzes synthesis of DNA. This enzyme has 5’ exonuclease activity, proofreading activity additional to DNA synthesis. DNA polymerase used in vitro conditions is isolated from the bacterium Thermus aquaticus which is highly thermostable and functions optimally at the temperature range of 750C-800C.

Restriction enzymes are widely used biological tools in Molecular biology. These are known as Molecular scissors. These enzymes have the ability to cut DNA recognizing a specific sequence. There are 4 types of restriction enzymes. Type II and Type IV are used in recombinant DNA technology. Restriction enzymes are named with the bacterium of which it has been isolated and corresponding number of the restriction site. Some recognition enzymes have a separate recognition site and a cleavage site while in some both are the same. Restriction enzymes with same cleavage and restriction site are called as Isochizomers. Type II enzymes are very stable which requires Mg2+ as cofactor and shows star activity ( change in specificity). Few examples for restriction enzymes are E co RI, Pvul I, Hae III, Alu I, Hpa I.

DNA ligases have the ability to join two DNA fragments which is called as ligation of DNA. Ligation depends on the type of ends to be joined whether sticky or blunt. This enzyme is usually isolated from E. coli cultures which are infected with T4 phage for genetic engineering. T4 ligase is capable of carrying out ligation invitro conditions. DNA ligase functions optimally at 370C.

Alkaline phosphatases are another group of enzymes used in Recombinant Technology. When plasmid vector for joining a foreign DNA fragment is treated with restriction enzymes, the cohesive ends of the broken plasmids instead of joining with foreign DNA join the cohesive end of the same DNA molecules and get re-circularized. To overcome this problem, restricted plasmid is treated with alkaline phosphatase which digests the 5’ phosphoryl group so that 5’ end of foreign DNA can covalently join to 3’ end of the plasmid. This enzyme is a diametric glycoprotein made up of two identical units and isolated from E.coli.

Reverse transcriptase enzyme is isolated from Avian Myeloblastosis virus which requires Mg or Mn for initiation of reverse transcription. To obtain DNA to clone a eukaryotic gene is more complicated because these contain regions called as introns that are non-coding regions. This enzyme cut introns out of mRNA and produce intron free DNA which is called c DNA.

Plasmid vectors are closed circular double stranded extra chromosomal units and widely used in recombinant DNA technology for carrying specific genes into an organism. Plasmids are used as they have the ability to self-replication. Plasmids used in genetic engineering carry specific genes called as marker genes which enable the selection of recombinant organisms in later stages. These marker genes include antibiotic resistant genes, genes for toxin production etc. These contain restriction sites which can be cleaved with restriction enzymes and can be replaced by gene of interest. Size of a plasmid ranges from 1-200kb. Example for plasmids is Ti plasmid isolated from Agrobacterium tumefaciens.

Apart from plasmids isolated from bacteria, viruses are also used as cloning vectors due to its ability of infection. Mostly, they are used as plant vectors. Cauliflower mosaic viruses, Gemini virus, Tobacco Mosaic Virus are some viruses used. As most of the viruses contain RNA as the genetic material, gene regulation functions through production of c DNA by reverse transcription. In contrast to plasmids, viral vectors can incorporate only a small fragment of DNA. Simian Virus, Vaccinia Virus and Retro Virus are some animal vectors. Vaccinia virus is used in vaccines to produce immunogenicity.

Cosmid is a hybrid between a plasmid and a bacteriophage DNA. These contain cos sequences of bacteriophages which directs insertion of DNA. Bacteriophages are viruses that infect bacteria. Cosmid is almost similar to a plasmid which has an origin of replication, restriction sites and marker genes.