title: The influence of nucleotide sequence and temperature on the activity of thermostable DNA polymerases.
abstract: Extension rates of a thermostable, deletion-mutant polymerase were measured from 50°C to 90°C using a fluorescence activity assay adapted for real-time PCR instruments. Substrates with a common hairpin (6-base loop and a 14-bp stem) were synthesized with different 10-base homopolymer tails. Rates for A, C, G, T, and 7-deaza-G incorporation at 75°C were 81, 150, 214, 46, and 120 seconds(-1). Rates for U were half as fast as T and did not increase with increasing concentration. Hairpin substrates with 25-base tails from 0% to 100% GC content had maximal extension rates near 60% GC and were predicted from the template sequence and mononucleotide incorporation rates to within 30% for most sequences. Addition of dimethyl sulfoxide at 7.5% increased rates to within 1% to 17% of prediction for templates with 40% to 90% GC. When secondary structure was designed into the template region, extension rates decreased. Oligonucleotide probes reduced extension rates by 65% (5'-3' exo-) and 70% (5'-3' exo+). When using a separate primer and a linear template to form a polymerase substrate, rates were dependent on both the primer melting temperature (Tm) and the annealing/extension temperature. Maximum rates were observed from Tm to Tm - 5°C with little extension by Tm + 5°C. Defining the influence of sequence and temperature on polymerase extension will enable more rapid and efficient PCR.
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Jesse L
authors.initials: JL
authors.lastname: Montgomery
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Nick
authors.initials: N
authors.lastname: Rejali
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, Utah. Electronic address: carl.wittwer@path.utah.edu.
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D055550:Protein Stability; D013696:Temperature; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase