COLD-PCR: improving the sensitivity of molecular diagnostics assays

Abstract

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold.

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.

Figure 2. Ice-coamplification at lower denaturation temperature-PCR protocol schematic

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].

Figure 3. Enrichment observed in amplicons produced by conventional PCR, full-COLD-PCR, fast-COLD-PCR and ice-COLD-PCR

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].

Figure 4. Enrichment potential for three PCR amplification strategies: conventional PCR, full-COLD-PCR and ice-COLD-PCR

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].

Figure 5. COLD-PCR improves mutation detection in downstream assays

(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].

Figure 6. High-resolution melt analysis of TP53 exon 6 amplification products produced via conventional and coamplification at lower denaturation temperature (COLD)-PCR

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].