Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations

Chiuan Chian Chiou*, Ji Dung Luo, Tai Long Chen

*此作品的通信作者

研究成果: 期刊稿件文章同行評審

26 引文 斯高帕斯(Scopus)

摘要

The detection of rare mutant DNA from a background of wild-type alleles usually requires laborious manipulations, such as restriction enzyme digestion and gel electrophoresis. Here, we describe a protocol for homogeneous detection of rare mutant DNA in a single tube. The protocol uses a peptide nucleic acid (PNA) as both PCR clamp and sensor probe. The PNA probe binds tightly to perfectly matched wild-type DNA template but not to mismatched mutant DNA sequences, which specifically inhibits the PCR amplification of wild-type alleles without interfering with the amplification of mutant DNA. A fluorescein tag (which undergoes fluorescence resonance energy transfer with the adjacent fluorophore of an anchor probe when both are annealed to the template DNA) also allows the PNA probe to generate unambiguous melting curves to detect mutant DNA during real-time fluorescent monitoring. The whole assay takes about only 1 h. This protocol has been used for detecting mutant K-ras DNA and could be applied to the detection of other rare mutant DNAs.

原文英語
頁(從 - 到)2604-2612
頁數9
期刊Nature Protocols
1
發行號6
DOIs
出版狀態已出版 - 01 2007

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