title: Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.
abstract: A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.
authors.affiliation: Molecular Sensing plc, Challeymead Business Park, Bradford Road, Melksham, Wiltshire SN12 8LH, UK. christy.waterfall@molecular-sensing.com
authors.forename: Christy M
authors.initials: CM
authors.lastname: Waterfall
authors.forename: Robert
authors.initials: R
authors.lastname: Eisenthal
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Cobb
mesh_terms: D000483:Alleles; D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers; D007700:Kinetics; D008962:Models, Theoretical; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D017931:DNA Primers; D019914:Taq Polymerase