Palindromes and staggered cleavage

This is the most common method, where due to the presence of a palindromic sequence, a restriction enzyme causes staggered cuts producing short complementary single stranded sticky ends (a palindromic sequence is one, which has complementary sequences at the two ends of a single strand, e.g. ATC—GAT). This can be illustrated using the example of enzyme EcoRl, which cuts the sequences at specific positions (Fig. 39.12). When another DNA molecule is similarly cut by the same enzyme, similar sticky ends having same sequences in the single stranded ends will be produced, so that when it is mixed with the previous molecule similarly treated, the two will anneal producing a chimeric DNA or chimeric plasmid, if plasmid DNA is involved (Fig. 39.13). Enzyme DNA ligase helps in joining the bonds at the cut ends of two molecules.

This technique has the advantage of regenerating two restriction sites (e.g., EcoRl site) in the chimeric DNA, so that the foreign DNA segment can be retrieved rather easily from the cloned copies of chimeric DNA by cleavage again with the same enzyme. There are also following disadvantages with this technique, (i) The two cleaved ends of a vector or of a foreign DNA may join end to end before getting inserted. Therefore, while isolating chimeric DNA, one will have to select chimeric molecules each having only a single insert. This can be achieved by separating molecules of different sizes by gel electrophoresis. (ii) The recognition site, particularly in the sequence to be cloned may not lie at a convenient position, so that sometimes only a part of the desired segment will be inserted.