Comparative analysis of dideoxy sequencing, the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

摘要: AIM: To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients. METHODS: Formalin-fixed, paraffin-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 CRC patients at Beijing Cancer Hospital. After extraction genome DNA FFPE samples, fragments contained codons 12 and 13 exon 2 amplified by polymerase chain reaction analyzed sequencing, StripAssay pyrosequencing. In addition, sensitivities 3 methods compared on serial dilutions (contents mutant DNA: 100%, 50%, 20%, 15%, 10%, 5%, 1%, 0%) A549 cell line (carrying codon Gly>Ser mutation) into wild-type (human normal intestinal mucosa). The results pyrosequencing Chromas Software, Collector PyroMarkTM Q24 system, respectively. RESULTS: Among patients, mutations identified 34%, 37% pyrosequencing, respectively. sensitivity was highest (1%), followed (5%), sequencing lowest (15%). Six different types found this study main Gly12Asp (GGT>GAT), Gly12Val (GGT>GTT) Gly13Asp (GGC>GAC). Thirty-three to have methods, a total 8 had conflicting methods: 4 not detected pyrosequencing; StripAssay; 1 confirmed StripAssay. these discordant results, consistent either or which indicated that accuracy high. CONCLUSION: Taking worldwide view reports our remains most popular method because its low cost high accuracy.