A Sensitive PCR-Based Method for Somatic Mutations Enrichment and Screening.

BACKGROUND

EGFR and KRAS are the most frequently mutated genes in lung cancers, occurring in about 60% of all cases. Mutation genes assay has emerged as a promising blood-based biomarker for monitoring cancer dynamics noninvasively. However, detection can be challenging in patients where plasma often contains low levels of tumor-derived DNA fragments.

METHODS

We have developed a nuclease-based enrichment assay for detecting mutant alleles. The procedure is based on Surveyor endonuclease cleaves mismatched DNA molecules, and these DNA fragments were enriched for mutation screening. We screened lung cancer specimens for mutations in exons 18 and 21 of EGFR, and the majority of activating mutations in lung cancer occur in codons 12 (G12X) and 13 (G13X) of exon 2 of the KRAS gene. The method screened all mutant genes with the same pair primers and three relevant TaqMan probes.

RESULTS

The method can effectively remove wild-type sequences and enrich mutation DNA, and the sensitivity detectable mutant allele frequencies (MAF) achieved 0.001%. The method increases the sensitivity and efficiency of mutation DNA for cancers screening. This highlights the importance of complex DNA variation like mutations in exon 21 of EGFR and exon 2 of the KRAS gene detected by the same probe.

CONCLUSION

We developed a simple and sensitive methodology for mutation gene screening. The method is a cost-effective and sensitive method for mutation DNA enrichment and detection.