COLD-HRM PCR versus conventional HRM PCR to detect the BRAF V600E mutation A real improvement?

摘要: To the Editor-in-Chief: In recent article by Mancini et al,1 authors demonstrated improved sensitivity of detection KRAS mutations and BRAF V600E mutation using coamplification at lower temperature PCR (COLD-PCR) in combination with high-resolution melting (HRM) instead conventional followed HRM. They reported an eightfold higher for (from 6.2% to 0.8%) a debatable fourfold 12.5% 6.2%, which would seem be twofold sensitivity), serial dilutions DNA from cell lines harboring (CCRF-CEM SKMel28) MCF-7 as wild-type control. Using HRM, we2 others, including authors,3 have previously limits less than 5% mutated background DNA, is more sensitive COLD-PCR assay described their present article. In addition, our system2 proved applicable FFPE material. With samples, spread curves increases due quality DNA; however, we were able distinguish samples containing DNA. We read great interest publications on advantages COLD-PCR. Indeed, used extensive optimization process try improve PCR. We designed new primer sets that yield short amplicons maximize effect caused mismatch heteroduplexes during COLD-PCR, taking into account single-nucleotide polymorphisms pseudogene gene. optimized reaction composition protocol. Unfortunately, yet not been (SKMel28) (human mononuclear DNA) could observe expected shift quantification cycle values preferential amplification minority alleles (unpublished data). could, confirm equal behavior 100% homozygously SKMel28 difference plots Thus, regard Figure 1 (right) study do understand different A (MCF-7 G (100% because no can formed these reactions T-A substitution should cause any alteration (in consideration hydrogen bonds). Thus, although has method ideal low-level increasing PCR-based assays, application Similar results shown Fadhil colleagues.4 intensive increased time necessary each run (approximately 6 hours) renders sophisticated but labor-intensive method. Although still being pursued laboratory, findings thus far are rather modest. request comment (1) (2) failure refer other studies,2 own work,3 showing limit HRM