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To the Editor:For minor and major DNA alleles, coamplification at lower denaturation temperature-PCR (COLD-PCR)1 selectively enriches minority alleles from mixtures of wild-type and mutation-containing sequences without a priori knowledge of the type or position of a mutation within a sequence (1). Since its inception (2), COLD-PCR has been employed in a variety of applications, including cancer, prenatal diagnostics, infectious diseases, and crop development (3). In COLD-PCR, mutation-containing sequences are enriched either because they have reduced melting temperatures ( T m) due to the type of mutations present (fast-COLD-PCR) or because they form heteroduplexes with wild-type DNA (full-COLD-PCR). In both cases the resulting T m difference is exploited for preferential denaturation and replication of mutated sequences by employing a critical denaturation temperature ( T c) for a given DNA sequence. The T c of an amplicon usually lies approximately 1 °C below the melting temperature ( T m) (4, 5). Because the critical denaturation temperature needs to be controlled precisely during PCR, a thermocycler with a high degree of accuracy and precision in temperature control is desirable, i.e., to within about ±0.2 °C. Unfortunately, this excludes several commonly used PCR machines. For example, the ECO real-time PCR machine from Illumina and the Lightcycler 1536 real-time PCR system from Roche are programmable to 1 °C increments, and the temperature of …

Enriching Mutant Sequences by Modulating the Denaturation Time during PCR