Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.
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.
Waterfall
Christy M
CM
Molecular Sensing plc, Challeymead Business Park, Bradford Road, Melksham, Wiltshire SN12 8LH, UK. christy.waterfall@molecular-sensing.com
Eisenthal
Robert
R
Cobb
Benjamin D
BD
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
D017931:DNA Primers; D019914:Taq Polymerase