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

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

26 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)2604-2612
Number of pages9
JournalNature Protocols
Volume1
Issue number6
DOIs
StatePublished - 01 2007

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