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.
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.
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.
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.
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