Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer
The selective step occurs only when a SuperSelective primer hybridizes to a DNA (-) template fragment present in the sample. Due to the small size of the foot sequence, the probability of initiation of a (+) amplicon is significantly greater if the target sequence of the foot in the (-) template fragment is a completely complementary mutant sequence, than if the target sequence of the foot in the (-) template fragment is a mismatched wild-type sequence. If (+) amplicon synthesis does occur, then the resulting (+) amplicon serves as a template for a conventional reverse primer, and is efficiently copied during the next thermal cycle, generating a (-) amplicon in which the complement of the unique bridge sequence that was present in SuperSelective primer is substituted for the intervening sequence that was present in the original (-) template fragment. As a result, in subsequent thermal cycles, the entire SuperSelective primer sequence is complementary to the (-) amplicon strands, and exponential amplification occurs efficiently, and can be followed in real-time.