Gene Cloning Assignment Help

Gene cloning involves the production and isolation of the DNA fragments to be cloned, its insertion into a suitable vector to obtain recombinant DNA, further introduction of the recombinant DNA into a suitable organism (the host) and then the selection of the transformed host cells which contain the desired gene/DNA fragment. The gene is then multiplied and expressed into the host.

Gene cloning utilizes certain biological products and biological agents for achieving its objectives. Four different types of enzymes are also used which are nucleases, ligases, polymerases, and DNA modifying enzymes.

a) Restriction endonucleases (also known as molecular scissors) to cut the DNA at specific sites.

b) A suitable DNA molecule capable of self replication in the selected host cell. This DNA molecule is called a vector and the DNA to be cloned is integrated into this vector.

c) DNA ligase (also known as molecular glue) to seal the nicks that remain in the recombinant DNA molecule.

d) A suitable organisms that serve as a host for propagation of the recombinant DNA, i.e. vector containing the DNA fragment to be cloned, viz., DNA insert

e) Reverse transcriptase is used to produce cDNA (complementary DNA) copies of, usually, mRNA that are used for creation of cDNA libraries.

f) Alkaline phosphatase for removing 5’-phophate from the DNA ends.

g) T4 polynucleotide kinase for addition of phosphate group to have an ends having a free 5’-OH.

h) SI nuclease for removal of single stranded protrusions from ends; both 3’- and 5’- extensions and removed.

i) The Klenow fragment of E. Coli DNA polymerase I to make the protruding ends double-stranded by extending the shorter segment.

j) Lambda exonuclease fro removal of nucleotides from the 5’-ends.

k) E.Coli exonuclease Bal31 for making DNA fragments blunt ends shorter from both its ends.

l) Linker and adapter oligonucleotide sequences for modification of the cut ends of DNA fragments.

The main topics which are considered in Recombinant DNA Technology are discussed in brief as follows:

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