Comparative analysis of pyrosequencing and QMC-PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection.

摘要: Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing the most versatile. We undertook a comparative these techniques. The are: To compare limit (LOD), mutations KRAS (codon 12/13 hotspot) BRAF (V600E were tested. DNA mixtures containing mutant alleles at frequency around 25%/12.5%/6%/3%/ 1.5%/0.8% analysed. mutation detection, 22 samples (nine quality from cell lines, 13 low formalin-fixed paraffin-embedded tissue) tested for three hotspots (codons 12/13, 61 146) two exon 11). HRM (codon12/13) (V600E) showed that 3% 1.5% respectively could be reliably detected whilst 6% each case. Of 110 tests performed on samples, 109 cases gave identical results. Two had previously been called as wild type by direct Sanger sequencing but found to both pyrosequencing. Both can detect small numbers although has lower detection. suitable use more sensitive than sequencing.