Application of COLD-PCR for improved detection of KRAS mutations in clinical samples

摘要: KRAS mutations have been detected in approximately 30% of all human tumors, and shown to predict response some targeted therapies. The most common mutation-detection strategy consists conventional PCR direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve sensitivity, we compared our method with the recently described co-amplification-at-lower denaturation-temperature (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, critical denaturation temperature lowered 80 degrees C (vs 94 PCR). COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens formalin-fixed paraffin-embedded (FFPE) tissue 30 solid tumors. Implementation straightforward required no additional cost for reagents instruments. specific reproducible. successfully that positive PCR, enhanced mutant-to-wild-type ratio >4.74-fold, increasing mutation 1.5%. enhancement inversely correlated tumor-cell percentage sample. conclusion, validated utility superior detecting variety hematopoietic tumors using either fixed, tissue.