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Rapid and Sensitive Detection of KRAS Mutation After fast-COLD-PCR Enrichment and High-resolution Melting Analysis

Song, Chen BSc; Milbury, Coren A. PhD; Li, Jin PhD; Liu, Pingfang PhD; Zhao, Meiping PhD; Makrigiorgos, Gerasimos Mike PhD

Diagnostic Molecular Pathology: June 2011 - Volume 20 - Issue 2 - p 81–89
doi: 10.1097/PDM.0b013e3181fde92f
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KRAS mutations exhibit significant predictive and prognostic value in cancer. Efficient, sensitive, and accurate molecular approaches are required to evaluate KRAS mutation status, even when mutant alleles are restricted to a small portion of a clinical sample, which otherwise contains wild-type alleles. We describe a highly sensitive method to detect KRAS mutations by high-resolution melting (HRM) analysis after mutation enrichment by fast-COLDpolymerase chain reaction (PCR). Using 10 ng of starting DNA and after fast-COLD-PCR of a 76-bp region containing KRAS codons 12 and 13; the amplicons undergo a nested conventional PCR reaction followed by HRM analysis. Samples exhibiting aberrant melting profiles are sequenced to identify mutation type and position. Serial dilution experiments indicate a sensitivity of approximately 0.3% mutant-to-wild type for HRM-based mutation detection and the ability to directly sequence mutation-containing samples. A number of lung adenocarcinoma specimens earlier characterized were screened. Fast-COLD-PCR-HRM analysis correctly identified KRAS mutations and also showed a previously undetected, low-level missense GGT>TTT complex mutation. On account of the short target regions and low requirement of starting DNA, this rapid, cost-effective, and sensitive fast-COLD-PCR-HRM approach is expected to find broad application for detecting low-abundance KRAS mutations in a wide range of clinical specimens.

*Department of Radiation Oncology, Division of Medical Physics and Biophysics

Division of Genome Stability and DNA Repair, Dana-Farber/Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

Supported by T32-CA009078 from the NCI (C.M.), by a student grant from the China Scholarship Council CSC 2009601213 (C.S.), by the JCRT Foundation (J.L.), and by NIH grants CA-138280 and CA-111994. The contents of this study are the responsibility of the authors and do not necessarily represent the official views of the NIH.

Chen Song and Coren A. Milbury contributed equally to this study.

The authors have no conflicts of interest (financial, professional, or personal) to declare. The study has not been published previously and is not being considered concurrently by another publication.

Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website, www.molecularpathology.com

Reprints: Gerasimos Mike Makrigiorgos, PhD, Dana-Farber/Brigham and Women's Cancer Center, Brigham and Women's Hospital, Level L2, Radiation Therapy, 75 Francis Street, Boston, MA 02115 (e-mail: mmakrigiorgos@partners.org); and Meiping Zhao, PhD, Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China (e-mail: mpzhao@pku.edu.cn).

© 2011 by Lippincott Williams & Wilkins.