Speaker: Jesse Montgomery. Advisor: Carl Wittwer, MD/PhD

Abstract:

Little is known about the limitations of PCR. Advancements in chemistry and instrumentation have increased the speed and specificity of the technique. Further improvements will be facilitated by a more complete understanding of the rates of the individual reactions. Reaction kinetics for DNA annealing, denaturation, and polymerase extension have been studied in a manner that is not applicable to PCR. Each is considered to occur at a constant rate during temperature holds within thermal cycling. As the speed of PCR increases, this approximation becomes inadequate. Transitions between temperatures dominate over hold times and reactions are concurrent and have fluctuating rates. We will develop a more complete description of PCR kinetics by measuring reaction rates over a range of temperatures using a stopped flow instrument with temperature control capabilities. Knowledge of the temperature dependence of reaction rates will enable improved thermal cycling and solution conditions for more rapid and efficient PCR.