PCR-based methods for the enrichment of minority alleles and mutations

Abstract

Background: The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing early and posttreatment tumor status in cancer patients. Over the past 2 decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze.

Content: Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknown mutations. Although there are several robust approaches for detecting known mutations within a high background of wild-type DNA, there are few techniques capable of enriching and detecting low-level unknown mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR amplicons containing unknown mutations at any position, such that they can be subsequently sequenced to identify the exact nucleotide change.

Summary: This review summarizes technologies available for detecting minority DNA mutations, placing an emphasis on newer methods that facilitate the enrichment of unknown low-level DNA variants such that the mutation can subsequently be sequenced. The enrichment of minority alleles is imperative in clinical and diagnostic applications, especially in those related to cancer detection, and continued technology development is warranted.

Fig. 1. Highly selective PCR-based methods for known and unknown mutation enhancement and identification

(A), Bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (bi-PAP-ASA) for high selectivity of known mutations (26). P* is a specifically designed oligonucleotide with a 3′-terminal blocker that is activated, but not extended, by pyrophosphorolysis. Downstream and upstream P* contain dideoxy C and G at the 3′ termini that are specific to the mutant but not the wild type. Efficient amplification of the mutant occurs after pyrophosphorolysis (to remove the 3′-terminal ddCMP) and polymerization. Inefficient amplification is denoted by the gray arrows. Nonspecific type I error amplification is rare; type II error is caused by serial mismatch phosphorolysis and misincorporation, which results in the exponential amplification of the mutated product and reduces selectivity. (B), Digital PCR for high selectivity of both known and unknown mutations (27). Genomic DNA is diluted to approximately 1–2 copies per well. The number (N) of required wells varies widely and depends on putative mutant and wild-type concentrations. PCR is performed on each sample well individually. PCR amplicons can be used in many downstream applications such as direct sequencing, pyrosequencing, TaqMan assays, and molecular beacons.

Fig. 2. COLD-PCR protocol (40). Two forms of COLD-PCR have been developed, Full COLD-PCR (A) and Fast COLD-PCR (B). An example protocol for a 167-bp TP53 amplicon is reviewed here

(A), Full COLD-PCR has the potential to enrich all possible mutations. Several preliminary rounds of conventional PCR enable an initial buildup of 1 or more target amplicons, then the cycling switches to COLD-PCR. After denaturation at 94 °C, the PCR amplicons are incubated (e.g., 70 °C for 2–8 min) for reannealing and cross-hybridization. Cross-hybridization of mutant and wild-type alleles forms a mismatch-containing structure (heteroduplex) that has a lower melting temperature than a fully-matched structure (homoduplex). The PCR temperature is next raised to the critical denaturation temperature (T_c) (e.g., 86.5 °C) to preferentially denature the heteroduplexed amplicons. The temperature is reduced for primer annealing (e.g., 55 °C) and then raised to 72 °C to extend the amplicon and preferentially amplify the mutation-containing alleles. (B), Fast COLD-PCR is a simpler cycling that can be performed to enrich for mutations that reduce the melting temperature of the wild-type amplicon. Using the mutant T_c, rather than the standard 94 °C denaturation temperature, preferentially denatures the lower-T_m allele. Fast COLD-PCR does not perform the 70 °C incubation step. Fast COLD-PCR amplification and enrichment begins earlier in the cycling than in full COLD-PCR, thus resulting in higher enrichment.

Fig. 3. COLD-PCR improves mutation detection in downstream assays (40)

(A), Sanger sequencing detects low-level mutations after COLD-PCR. The HCC2218 cell line (TP53 exon 8; 14516 C>T) was diluted in wild-type DNA. Sanger sequencing was performed on products amplified by both conventional (upper panel) and COLD (lower panel; T_c 86.5 °C) PCR. COLD-PCR sequencing exhibits enrichment of the mutated allele. (B), COLD-PCR improves detection via pyrosequencing. DNA from cell line A549 was diluted 33-fold into wild-type DNA; a 98-bp K-ras exon 2 segment was amplified by both COLD-PCR (lower panel; T_c 80 °C) and conventional PCR (upper panel), followed by pyrosequencing. The G>A mutation of the A549 cell line was visible only when COLD-PCR was applied. (C), COLD-PCR improves the sensitivity of MALDI-TOF genotyping technologies. Fast COLD-PCR (T_c 83.5 °C) was used to amplify an 87-bp fragment in plasma-circulating DNA (hotspot mutation TP53 exon 8, codon 273). Amplicons were genotyped using MALDI-TOF. The G>A mutation was detectable in COLD-PCR amplicons (lower panel); however, it was undetectable in conventional PCR amplicons (upper panel). (D), COLD-PCR improves the sensitivity of TaqMan genotyping technologies. Serial dilutions of the H1975 cell line (containing T790M mutation of EGFR exon 20) in wild-type DNA were screened with conventional and COLD-PCR TaqMan genotyping for T790M mutation. Upper panel: conventional PCR TaqMan genotyping for T790M mutation; lower panel: COLD-PCR TaqMan genotyping for T790M mutation.