1 - The Use of Cosolvents to Enhance Amplification by the Polymerase Chain Reaction

Abstract

This chapter presents a study to show how cosolvents influence the polymerase chain reaction (PCR) by determining their effects on template melting properties and on the activity and thermostability of Taq DNA polymerase. In this study, Taq DNA polymerase activity was assayed by determining the level of incorporation of labeled nucleotide monophosphate into an activated salmon sperm DNA template. The thermostability of Taq DNA polymerase was determined by steady-state thermal inactivation performed in a constant-temperature waterbath at 95°C or 97.5°C. Two types of effects were seen on Taq DNA polymerase activity. A decrease in polymerase activity at both 60°C and 74°C was exhibited in the presence of DMSO and glycerol, which is characteristic of enzyme inhibition. The data from the study showed that the use of glycerol and formamide in a PCR facilitated both amplification at lower denaturation temperatures and amplification from templates with high G + C content. The ability to lower melting temperature by the use of cosolvents may allow the use of lower denaturation temperatures in a PCR, but may also affect the annealing temperature of primers.