A continuous fluorescence assay for polymerase activity

Little is known about the kinetic limitations of the polymerase chain reaction (PCR). Advancements in chemistry and instrumentation have increased its speed and specificity. Further improvements will be facilitated by a more complete understanding of the rates of the individual reactions that comprise PCR. A continuous fluorescent assay is developed to study DNA polymerase extension. Nucleotide incorporation is monitored with noncovalent DNA dyes using a defined hairpin template. The extension rate is measured in nucleotides incorporated per second per molecule of polymerase and has greater relevance to PCR than traditional activity methods. This assay was developed and validated on a stopped-flow instrument and subsequently adapted for real-time PCR instruments to extend its utility to any laboratory setting. The influences of a variety of buffer components were determined and optimal conditions for fast polymerase extension are recommended. The incorporation rates of each nucleotide were determined and extension was found to depend on template sequence. When DMSO was included in the reaction to reduce inhibition from secondary structure, extension rates of random sequences were closely approximated by their base composition. Extension rates as a function of temperature were determined and were applied to a kinetic model. This model accounts for extension during temperature transitions and more accurately portrays fast PCR with rapid thermal cycling. A complete model of PCR based on differential equations derived from mass action equations is provided. This can be used to incorporate experimentally derived parameters obtained for the other reactions of PCR. Knowledge of the temperature and chemistry dependence of reaction rates will enable improved thermal cycling and solution conditions for more rapid and efficient PCR.