Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations
| العنوان: | Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations |
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| المؤلفون: | Tai-Long Chen, Ji-Dung Luo, Chiuan-Chian Chiou |
| المصدر: | Nature Protocols. 1:2604-2612 |
| بيانات النشر: | Springer Science and Business Media LLC, 2006. |
| سنة النشر: | 2006 |
| مصطلحات موضوعية: | Peptide Nucleic Acids, Gel electrophoresis, Peptide nucleic acid, DNA Mutational Analysis, Mutant, Molecular Probe Techniques, Biology, Polymerase Chain Reaction, Molecular biology, General Biochemistry, Genetics and Molecular Biology, DNA sequencing, law.invention, chemistry.chemical_compound, Förster resonance energy transfer, chemistry, law, Polymerase chain reaction, DNA |
| الوصف: | 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. |
| تدمد: | 1750-2799 1754-2189 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5ecb8ae6a8e969452fe372a51a399334 https://doi.org/10.1038/nprot.2006.428 |
| حقوق: | CLOSED |
| رقم الأكسشن: | edsair.doi.dedup.....5ecb8ae6a8e969452fe372a51a399334 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 17502799 17542189 |
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