Joining DNA Molecules

Joining DNA Molecules :

Having described the methods available for cutting DNA molecules we must consider the ways in which DNA fragments can be joined to create artificially recombinant molecules. There are currently three methods for joining DNA fragments in vitro. The first of these capitalizes on the ability of DNA ligase to join covalently the annealed cohesive ends produced by certain restriction enzymes. The second depends upon the ability of DNA ligase from phage T4-infected E. coil to catalyse the formation of phosphod
iester bonds between blunt-ended fragments. The third utilizes the enzyme terminal deoxy nucleotidyl-transferase to synthesize homopolymeric 3 ‘-single-stranded tails at the ends of fragments. We can now look at these three methods a little more deeply.

                      DNA Ligase
                             E. coil and phage T4 produce an enzyme, DNA ligase, which seals single. stranded nicks between adjacent nucleotides in a duplex DNA chain (Olivera et al. 1968, Gumport & Lehman 1971). Although the reactions
catalysed by the enzymes of E. coil and T4-infccted E. coil are very similar, they differ in their cofactor requirements. The T4 enzyme requires ATP,