Insertion Of The Gene Into A Suitable Vector
a) Homopolymer tailing
This method of joining DNA molecules makes use of the annealing of complementary homopolymer sequences. Thus by adding oligo (dA) sequences to the 3’ ends of one population of DNA molecules and oligo (dT) blocks to the 3’ ends of another population, the two types of molecules can be annealed to form mixed dimeric circles.
An enzyme purified from calf thymus, terminal deoynucleotidyl transferase, provides the means by which homopolymeric extensions can be synthesized.
b) Ligation cohesive termini
When both the vector and the DNA insert are cut with the same restriction enzymes, they have completely matched cohesive ends, opened up vector and DNA are mixed under annealing conditions, allowing them to pair between cohesive ends of vector and the DNA insert. The nicks remaining after annealing are sealed, by DNA ligase.
c) Blunt end ligation (no linker is used).
High concentration of both, the enzyme, the vector and insert DNA are required for the joining of blunt-ended vector and DNA insert by T4DNA ligase, as high concentration increases the chances of blunt ends coming together in correct way.
d) Linker molecules.
If the vector and the DNA insert have one compatible cohesive end and the other the flush end, their cohesive end pair together when mixed under annealing conditions. The T4 ligase seals the nicks at the cohesive ends joining the blunt ends as well. Only recombinant DNA molecules are produced by this process.
Four different types of enzymes are used viz. nucleases, ligases, polymerases, and certain DNA modifying enzymes.
- For the production and isolation of the DNA fragment one of the following methods may be used:
- For the insertion of the gene into a suitable vector either of these methods are used:
- For further introduction of the recombinant DNA molecule into the host cell one of these methods is suitable:
- The end process of screening for the desired clones is carried out in one of the following manner:
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