Figure 1. Coamplification at lower denaturation temperature-PCR protocol.

Two original forms of COLD-PCR were developed as full-COLD-PCR (A) and fast-COLD-PCR (B). (A) Full-COLD-PCR has the potential to enrich all possible mutations. Several preliminary rounds of conventional PCR enable an initial increase of the target amplicon(s). After denaturation at approximately 95.0°C (or as defined by the polymerase system), the PCR amplicon(s) are incubated (e.g., 70.0°C for 2–8 min) for re-annealing and hybridization. Hybridization of mutant and wild-type alleles forms heteroduplexed molecules (mismatch-containing) that possess a lower T_m than homoduplexed molecules. The PCR temperature is subsequently increased to the T_c (e.g., T_c = 86.5°C) to preferentially denature the heteroduplexed amplicons. The temperature is reduced for primer annealing (e.g., 55.0°C), and then increased to 72.0°C for primer extension, thus preferentially amplifying the mutation-containing alleles. (B) Fast-COLD-PCR can be performed to enrich mutations with melting temperatures lower than the wild-type amplicon. Denaturation at the T_c (rather than the standard 95.0°C) preferentially denatures the strands containing the lower T_m allele; this generates single-stranded DNA for primer annealing and extension.

COLD: Coamplification at lower denaturation temperature; dsDNA: Double-stranded DNA; T_c: Critical denaturation temperature; T_m: Melting temperature.