Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3°C. This precludes simultaneous mutation enrichment in sequences of substantially different melting-temperature (Tm) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT-COLD-PCR) approach that reduces this obstacle.
Thermo-cycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR are described. This approach enables enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation is provided for KRAS and TP53 exons 6–9 using dilutions of mutated DNA, clinical cancer samples and plasma-circulating DNA.
A single thermocycling program with a denaturation-temperature window of 2.5–3.0°C enriches mutations in all DNA amplicons simultaneously, despite their different Tms. Mutation enrichments of 6–9-fold were obtained using TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other two forms of COLD-PCR, fast-COLD-PCR and ice-COLD-PCR.
Low-level mutations in diverse amplicons with different Tm can be mutation-enriched via TT-COLD-PCR provided that their Tms fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons, and increases significantly the versatility of COLD-PCR.