The primary goal of this work is to explore the application of mismatch binding proteins derived from the hyperthermophilic eubacteria Aquifex pyrophilus (Apy) and Thermotoga maritima (Tma) to improve detection of mutations and polymorphisms through enhanced allele-specific PCR amplification. The basic technology is a mismatch-specific TaqMan PCR. A TaqMan oligonucleotide (oligo) is an unextendable oligo which binds to a template, thermostable mismatch-binding proteins will bind to the mismatch between the TaqMan oligo and template, thermostable mismatch-binding proteins will bind to the mismatch and prevent DNA polymerization. Thus a primer matching a mutant allele will select against amplification product, the same selection takes place at each cycle, permitting geometric selection. The hypothesis is that this simple closed tube technology for detecting mutant alleles in a vast excess of normal alleles will have important applications in the study of cancer and cancer epidemiology. The specificity and rate of mismatch binding protein-heteroduplex complex formation and dissociation at mismatches and bulge loops will be determined to explain the competition between DNA polymerization and TaqMan inhibition. DNA polymerization through matched and mismatched TaqMan primer-template complexes will be investigated to optimize the choice of DNA polymerases, mismatch binding proteins and TaqMan oligo design in terms of both PCR sensitivity and allele specificity. The effect of mismatch binding proteins on primer-detected allele-specific PCR will be investigated primarily for their potential to improve PCR primer specificity in general. Their effect in PCR fidelity will be investigated to determine the upper limit on specificity of mismatch-specific TaqMan PCR and to improve PCR fidelity in general, with applications to reduce PCR stuttering and faithful PCR cloning.
