High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancerReport as inadecuate




High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer - Download this document for free, or read online. Document in PDF available to download.

BMC Cancer

, 6:295

First Online: 21 December 2006Received: 27 September 2006Accepted: 21 December 2006DOI: 10.1186-1471-2407-6-295

Cite this article as: Krypuy, M., Newnham, G.M., Thomas, D.M. et al. BMC Cancer 2006 6: 295. doi:10.1186-1471-2407-6-295

Abstract

BackgroundThe development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis HRM to screen for KRAS mutations in clinical cancer samples. In non-small cell lung cancer, KRAS mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important.

MethodsWe developed a high-resolution melting HRM assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for KRAS mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in EGFR exons 18–21.

ResultsKnown KRAS mutations in cell lines A549, HCT116 and RPMI8226 were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5–6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had KRAS mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in KRAS and EGFR were mutually exclusive.

ConclusionHRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2407-6-295 contains supplementary material, which is available to authorized users.

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Author: Michael Krypuy - Genni M Newnham - David M Thomas - Matthew Conron - Alexander Dobrovic

Source: https://link.springer.com/







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