Answer any two questions (restrict each answer within 200 words):
Give a detailed account of the enzymes used in recombinant DNA technology.

Explanation

The enzymes involved in recombinant DNA technology are:
1. Nucleases These are the enzymes used for cutting of DNA at specific locations during rDNA technology. They are nucleic acid-digesting enzymes that break phosphodiester bonds,. e.g. ribonuclease, DNA nuclease, etc. These are of following two types:
(i) Exonucleases They remove nucleotides from the ends of the DNA (either 5. or 3.) in one strand of duplex. Based on their action on the strand, they are exo` (3. to 5.) and " exo " (5. to 3.)` type.
(ii) Endonucleases They make cut at specific position within DNA. They can make cut on linear and circular strands also, so as to generate polynucleotide fragments of variable size.
The most important and widely studied endonucleases are restriction endonucleases.
Restriction Endonucleases (RE)
These enzymes recognize specific recognition sequences on DNA and make the cut either within that sequence only, or at a variable distance from that sequence. The site where they cleave or cut the DNA is called recognition site. In rDNA technology, class-II REs are used.
2. Polymerases These enzymes catalyse the process of copying the nucleic acid molecules. These are of following types:
(i) RNA polymerases These are DNA dependent RNA polymerase.
(ii) DNA polymerases They copy DNA strand into another complementary strand through replication. These are also the DNA dependent enzymes.
(iii) Reverse transcriptases They catalyse the synthesis of complementary DNA (cDNA) strand on RNA template. These are found in retroviruses and are RNA dependent DNA polymerase.
(iv) RNA polymerases They catalyse the process of strand copying of DNA into RNA through transcription.
3. DNA ligases These enzymes help to join or seal 3.-OH end of one nucleic acid fragment with the 5.-phosphate end of another nucleic acid fragment by forming a phosphodiester bond. As they are able to stick DNA fragments together, they are known as molecular glue. The widely used ligase enzyme is T4 DNA ligase. It is purified from E. coli cells that are infected. by T4 bacteriophages.
4. End modifying enzymes These enzymes have the ability to modify the ends or termini of DNA molecules, e.g. alkaline phosphatase, polynucleotide kinase and terminal transferase.