Studies On The Establishment And Clinical Application Of Detection For Free DNA KRAS Mutation In The Peripheral Blood

With the advent of personalised medicine, there is a compelling need for rapid and accurate methods for detection of nucleic acid sequence changes in clinical specimens. An ideal technology should be sensitive enough to accommodate significant amounts of stromal and normal cell contamination, robust and simple enough to be readily implemented in a diagnostic laboratory, rapid enough to provide important therapeutic information in a clinical timeframe, and cost-efficient. High resolution melting (HRM) is an emerging technique for detection of nucleic acid sequence variation that has enormous potential to meet these clinical demands. We describe here the application of high resolution melting analysis (HRM) to screen for KRAS mutations of codon 12 and 13 mutations in tissue and plasma.We developed 3 different high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using saturated fluorescent dye. We tested cell line dilutions with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using the 3 different HRM assay. The COLD-PCR-based unlabled-probe HRM was most sensitive in detecting mutations and was able to detect as little as 0.1% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could discriminate only 20% mutant cell ratios.The shorter 59 bp amplicon was more sensitive than the 163 bp amplicon and was able to detect as little as 3% of mutant cell lines DNA, so was unlabled-probe HRM.We then screened for KRAS mutations in 43 pancreatic cancer tissues using unlabled-probe HRM. Simultaneously, we tested the specimens using Sanger sequencing. T-A DNA cloning and sequencing was applied to the differences. 20 (46.5%) of the 43 pancreatic cancer tissues had KRAS mutations detected by Unlabled-probe analysis, whereas 13 (30.2%) only had KRAS mutations detected by Sanger sequencing. The results of T-A DNA cloning and sequencing matched those of unlabled-probe HRM.The COLD-PCR-based unlabled-probe HRM was then used to screen for KRAS mutations in plasma DNA of 36 patients with pancreatic cancer,23 with gastrointestinal disease and 24 healthy individuals.26(72.2%) of the 36 pancreatic cancer plasma showed KRAS mutations. No mutation was found in the 47 samples of disease and normal controls. The detection sensitivity, specificity, and accuracy of COLD-PCR-based unlabeled probe melting analysis for KRAS mutations in plasma DNA samples were 80.6%,87.5% and 83.3%, respectively.The summary of our approach of noninvasive, convenient, extremely high-sensitive KRAS mutation analysis in plasma might provide diagnostic and nformation to clinicians.