A non-isotopic method for the determination of activity of the thermostable NAD-dependent DNA ligase from Thermus thermophilus HB8.

ABSTRACT

A simple and accurate method for determination of enzymatic activity of the NAD-dependent DNA ligase of Thermus thermophilus HB8 has been developed that requires no radiolabeled substrates. lambda-DNA digested with BstEII provides two substrate DNA molecules (fragments 1 and 4) containing 12 base pair cohesive ends that are stably annealed at the assay temperature of 45 degrees C. One cohesive end unit is defined as the amount of enzyme required to achieve 50% ligation of fragment 1 in 15 min at 45 degrees C. Percent ligation is determined by analysis of reaction products, produced in reactions containing serial dilutions of enzyme, separated by agarose gel electrophoresis and photographed using a digital imaging device. Imaging software quantifies the amounts of fragment 1 and non-substrate fragment 7 present in the each lane (reaction). The latter is used to normalize the amount of fragment 1. This normalization process corrects for variations in sample loading, electrophoretic artifacts, and optical distortion of the gel image. A negative control containing no enzyme allows calculation of percent substrate ligated into product. Unit activity is then calculated from a dose-response curve in which percent of fragment 1 ligated is plotted against the log(10) of the enzyme dilution factor.