Multiple heat pulses during PCR extension enabling amplification of GC-rich sequences and reducing amplification bias.

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele.