Development and clinical application of highly sensitive detection method (AL-COLD-PCR-SSCP) for somatic mutations in cancer

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In cancer, low level mutations cannot easily detected because of the much more DNA amounts of a wild-type background. Therefore, mutation enrichment technique is necessary. We tried to develop a novel detection method, Adapter ligation COLD-PCR (AL-COLD-PCR), however, we could not establish the method. Next, we investigated COLD-PCR-HRM (high resolution melting) method. Genomic DNA purified from cancer cell lines with K-ras mutation was diluted by wild-type DNA from other cell lines. We compared the detection sensitivity of K-ras mutations by COLD-PCR and conventional PCR, by use of the following HRM analysis. Full COLD-PCR can detect with higher sensitivity than conventional PCR, in coupling with HRM analysis. We applied this COLD -PCR-HRM analysis on somatic mutation detection in clinical specimens of colorectal cancer and lung cancer, and most of the mutation alleles could be detected. In conclusion, COLD-PCR-HRM could be utilized in mutation enrichment and detection of low-level mutations or mutants in cancer.