Ligation: Joining DNA molecules tog

Ligation: Joining DNA molecules togetherThe final step in the construction of a recombinant DNA molecule is a joining togetherof the vector molecule and the DNA to be cloned (Figure 4.19). This process is referredto as ligation, and the enzyme that catalyses the reaction is called DNA ligase.4.3.1 The mode of action of DNA ligaseAll living cells produce DNA ligases, but the enzyme used in genetic engineering is usu￾ally purified from E. coli bacteria that have been infected with T4 phage. Within thecell, the enzyme carries out the very important function of repairing any discontinuitiesthat may arise in one of the strands of a double-stranded molecule (see Figure 4.4a).A discontinuity is quite simply a position where a phosphodiester bond between adja￾cent nucleotides is missing (contrast this with a nick, where one or more nucleotides areabsent). Although discontinuities may arise by a chance breakage of the cell’s DNAmolecules, they are also a natural result of processes such as DNA replication andrecombination. Ligases therefore play several vital roles in the cell.In the test tube, purified DNA ligases not only repair single-strand discontinuitiesbut also join together individual DNA molecules, or the two ends of the same molecule.The chemical reaction involved in ligating two molecules is exactly the same as discon￾tinuity repair, except that two phosphodiester bonds must be made, one for each strand(Figure 4.20).