Adding poly dA at the 3' ends of the vector and poly dT at the 3' ends of DNA clone
In this method DNA can be cut at the desired position both in the vector and in the clone, without staggered cleavage. Using precursor dATP, poly dA is added at both the 3' cut ends of the vector with the help of the enzyme terminal transferase. Similarly, using precursor dTTP, poly dT is added at both the 3' cut ends of the DNA sequence to be cloned. The vector and clone can then the joined by annealing the poly dA with poly dT tails and then ligating them using DNA ligase enzyme (Fig. 39.14). In this technique, there is no chance of reannealing between the two cut ends of the same DNA molecule, so that one of the disadvantages of the first method has been overcome in this case. However, in this technique it will not be easy to retrieve the cloned DNA, because the recognition site of the enzyme due to insertion of poly dA and poly dT has been lost in this case. But if poly dG and poly dC are used instead (which serve the same purpose), cloned sequence can be retrieved, since poly dG : poly dC regenerate the recognition site for the enzyme Pstl or Small (Table 39.1).