Fred Kramer - SuperSelective PCR Primers for the Detection of Rare Mutant Cancer Cells

"SuperSelective" primers, by virtue of their unique design, enable only a few molecules of a mutant sequence to generate amplicons in conventional, real-time PCR assays without interference from extremely abundant wild-type molecules, even if the only difference between the mutant sequence and the wild-type sequence is a single-nucleotide polymorphism. The basis of this extraordinary selectivity is two-fold: (1) short, perfectly complementary mutant hybrids formed by the primers are thermodynamically more likely to be present at any given moment than the even shorter, mismatched wild-type hybrids; and (2) because the mean persistence time of mismatched wild-type hybrids (measured in milliseconds) is significantly shorter than the mean persistence time of perfectly complementary mutant hybrids, DNA polymerase molecules are kinetically more likely to encounter mutant hybrids before those hybrids dissociate, compared to the probability of their encountering the shorter-lived wild-type hybrids before they dissociate. Consequently, as few as 10 mutant molecules can routinely be distinguished and quantitated in samples containing 1,000,000 wild-type molecules.