ArticleNucleic acids research2006
Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe.
Article in Nucleic acids research, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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Who cites it
31 citing papers in PubMed, 65 citations in OpenAlex.
- Trial
- Molecular Characterization and Its Clinical Application of GNAS Variants in Intramuscular Myxoma.Cancer medicine · 2026Article
- Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis.Biochemical genetics · 2024Article
- Design of Oligonucleotides for Allele-Specific Amplification Based on PCR and Isothermal Techniques.Methods in molecular biology (Clifton, N.J.) · 2022Article
- A Pterin-FAM-TAMRA Tri-color Fluorescence Biosensor to Detect the Level of KRAS Point Mutation.Analytical sciences : the international journal of the Japan Society for Analytical Chemistry · 2020Article
- PNA Clamping in Nucleic Acid Amplification Protocols to Detect Single Nucleotide Mutations Related to Cancer.Molecules (Basel, Switzerland) · 2020Review
- Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by DeepMelt Assay.Journal of clinical microbiology · 2018Article
- Article
- Detection of Rare Somatic GNAS Mutation in McCune-Albright Syndrome Using a Novel Peptide Nucleic Acid Probe in a Single Tube.Molecules (Basel, Switzerland) · 2017Article
- Enzyme-mediated single-nucleotide variation detection at room temperature with high discrimination factor.Chemical science · 2015Article
- Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations.PloS one · 2015Article
- Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.Molecular diagnosis & therapy · 2014Article
- Kinetic hairpin oligonucleotide blockers for selective amplification of rare mutations.Scientific reports · 2014Article
- KRAS genotypic changes of circulating tumor cells during treatment of patients with metastatic colorectal cancer.PloS one · 2014Article
- Real-time bidirectional pyrophosphorolysis-activated polymerization for quantitative detection of somatic mutations.PloS one · 2014Article
- Molecular diagnosis of response to neoadjuvant chemoradiation therapy in patients with locally advanced rectal cancer.Journal of the American College of Surgeons · 2011Article
- COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma.BMC clinical pathology · 2010Article
- Detection of low-level KRAS mutations using PNA-mediated asymmetric PCR clamping and melting curve analysis with unlabeled probes.The Journal of molecular diagnostics : JMD · 2010Article
- Mutation detection of epidermal growth factor receptor and KRAS genes using the smart amplification process version 2 from formalin-fixed, paraffin-embedded lung cancer tissue.The Journal of molecular diagnostics : JMD · 2010Article
- An electrochemical sensor for single nucleotide polymorphism detection in serum based on a triple-stem DNA probe.Journal of the American Chemical Society · 2009Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 (T(m)) shift in a range of 9 to 16 degrees C. An extension temperature of 60 degrees 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.
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