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.

Data are presented for approximately 3% mutation abundance of T_m-increasing, T_m-equivalent and T_m-reducing mutations.
COLD: Coamplification at lower denaturation temperature; T_m: Melting temperature. Reproduced with permission from [28].

(A) Sanger sequencing detects low-abundance mutations after COLD-PCR. A 10% abundance of a C>T mutation in WT DNA was amplified by both conventional PCR and COLD-PCR. Sanger sequence chromatograms were evaluated for both approaches, respectively (conventional PCR is presented in the upper panel; COLD-PCR is presented in the lower panel); an approximate sixfold mutation enrichment by COLD-PCR is evident. (B) COLD-PCR improves detection via pyrosequencing. A 33-fold dilution of a G>A mutation in WT DNA was amplified by both conventional and COLD-PCR. The mutant is only visible in the COLD-PCR amplicons. (C) COLD-PCR improves the sensitivity of MALDI-TOF genotyping technologies. A G>A mutation was amplified by both conventional and COLD-PCR, and amplicons were genotyped using MALDI-TOF. The G>A mutation was undetectable in conventional PCR amplicons (upper panel); however, it was detectable in COLD-PCR amplicons (lower panel). (D) COLD-PCR improves the sensitivity of TaqMan^® genotyping technologies [35]. Serial dilutions of the human H1975 cell line (containing the T790M mutation in EGF receptor exon 20) in WT DNA were screened with conventional and COLD-PCR TaqMan genotyping for the T790M mutation. Conventional PCR TaqMan genotyping for the T790M mutation is presented in the upper panel; COLD-PCR TaqMan genotyping for the T790M mutation is presented in the lower panel.
COLD: Coamplification at lower denaturation temperature; WT: Wild-type.
Data taken from [24,35].

Ice-COLD-PCR has the potential to enrich all possible mutations and also provides a higher enrichment than full-COLD-PCR. Ice-COLD-PCR is performed in a nested format here (i.e., using a larger PCR amplicon as a template). An initial few rounds of nested conventional PCR are first applied to enable build-up of the target amplicon(s). After denaturation at approximately 98.0°C (or as defined by the polymerase system), the PCR amplicon(s) and the oligonucleotide reference sequence are hybridized at 70.0°C. Mutant-containing heteroduplexed molecules (mismatch-containing) will be denatured at the T_c, and preferentially enriched throughout the course of ice-COLD-PCR.
COLD: Coamplification at lower denaturation temperature; RS: Reference sequence; T_c: Critical denuration temperature; WT: Wild-type. Data from [28].

Enrichment is inversely related to initial mutation abundance for a melting temperature-equivalent (G>C) mutation, whereas higher enrichment potential is exhibited in lower mutation abundances. COLD: Coamplification at lower denaturation temperature.
Data taken from [28].

The amplicons were produced from genomic DNA serial dilutions of the human cell line SNU-182 (c.644G>T, p.S215I) in WT DNA. While the 2% mutant abundance is the lower limit of detection in the conventional PCR amplicons, COLD-PCR enriches the mutant fraction so that an initial mutant abundance as low as 0.1% can now be detected.
WT: Wild-type.
Data taken from [37].