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Nucleic Acids Research 2006 34(2):e12; doi:10.1093/nar/gnj008
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Published online 23 January 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe

Ji-Dung Luo1, Err-Cheng Chan1, Chun-Liang Shih1, Tai-Long Chen1, Ying Liang2, Tsann-Long Hwang2 and Chiuan-Chian Chiou1,*

1Department of Medical Biotechnology and Laboratory Science, Chang Gung University Taoyuan 333, Taiwan 2Department of Surgery, Chang Gung Memorial Hospital Taoyuan 333, Taiwan

*To whom correspondence should be addressed: Tel: +886 3 2118800, ext. 5204; Fax: +886 3 2118035; Email: ccchiou{at}mail.cgu.edu.tw

Received September 3, 2005. Revised December 5, 2005. Accepted December 28, 2005.

The major problem of using somatic mutations as markers of malignancy is that the clinical samples are frequently containing a trace amounts of mutant allele in a large excess of wild-type DNA. Most methods developed thus far for the purpose of tickling this difficult problem require multiple procedural steps that are laborious. We report herein the development of a rapid and simple protocol for detecting a trace amounts of mutant K-ras in a single tube, one-step format. In a capillary PCR, a 17mer peptide nucleic acid (PNA) complementary to the wild-type sequence and spanning codons 12 and 13 of the K-ras oncogene was used to clamp-PCR for wild-type, but not mutant alleles. The designated PNA was labeled with a fluorescent dye for use as a sensor probe, which differentiated all 12 possible mutations from the wild-type by a melting temperature (Tm) shift in a range of 9 to 16°C. An extension temperature of 60°C and an opposite primer 97 nt away from the PNA were required to obtain full suppression of wild-type PCR. After optimization, the reaction detected mutant templates in a ratio of 1:10 000 wild-type alleles. Using this newly devised protocol, we have been able to detect 19 mutants in a group of 24 serum samples obtained from patients with pancreatic cancer. Taken together, our data suggest that this newly devised protocol can serve as an useful tool for cancer screening as well as in the detection of rare mutation in many diseases.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint first authors


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