DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples

摘要: BACKGROUND: Mutation scanning provides the simplest, lowest-cost method for identifying DNA variations on single PCR amplicons, and it may be performed before sequencing to avoid screening of noninformative wild-type samples. High-resolution melting (HRM) is most commonly used mutation scanning. With PCR-HRM, however, mutations less abundant than approximately 3%–10% that can still clinically significant often missed. Therefore, enhancing HRM detection sensitivity important its clinical application. METHODS: We serial dilution cell lines containing TP53 exon 8 demonstrate improvement in conventional-PCR-HRM presence DMSO. also conducted coamplification at lower denaturation temperature (COLD)-PCR with an extra step cross-hybridization, followed by preferential amplification optimized critical ( full -COLD-PCR), further enrich low-level or without DMSO, we droplet-digital derive optimal conditions enrichment. Both conventional PCR-HRM -COLD-PCR-HRM DMSO were cancer samples known myelodysplastic syndrome unknown mutations. Mutations other genes examined. RESULTS: The increases 2- 5-fold depending type sequence context, typically detect abundance 1%. When enrichment applied during -COLD-PCR 0.2%–0.3% detected. CONCLUSIONS: improves saturating dyes. used, DMSO-HRM, overall about 20-fold compared PCR-HRM.