IMPROVING THE FIDELITY OF THERMUS-THERMOPHILUS DNA-LIGASE

The DNA ligase from Thermus thermophilus (Tth DNA ligase) seals single -strand breaks (nicks) in DNA duplex substrates. The specificity and t hermostability of this enzyme are exploited-in the ligase chain reacti on (LCR) and ligase detection reaction (LDR) to distinguish single bas e mutations associated with genetic diseases. Herein, we describe a qu antitative assay using fluorescently labeled substrates to study the f idelity of Tth DNA ligase. The enzyme exhibits significantly greater d iscrimination against all single base mismatches on the 3'-side of the nick in comparison with those an the 5'-side of the nick. Among all 1 2 possible single base pair mismatches on the 3'-side of the nick, onl y T-G and G-T mismatches generated a quantifiable level of ligation pr oducts after 23 h incubation. The high fidelity of Tth DNA ligase can be improved further by introducing a mismatched base or a universal nu cleoside analog at the third position of the discriminating oligonucle otide. Finally, two mutant Tth DNA ligases, K294R and K294P, were foun d to have increased fidelity using this assay.