Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer
| Autor: | V. P. Shelepov, Anatoly V. Lichtenstein, Natalia N. Mazurenko, Valentina N. Kondratova, Olga Y. Susova, I. V. Botezatu, Irina V. Tsyganova |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Adult
Male Mutant DNA Mutational Analysis Biophysics medicine.disease_cause 01 natural sciences Biochemistry Polymerase Chain Reaction law.invention Proto-Oncogene Proteins p21(ras) 03 medical and health sciences Nucleic acid thermodynamics symbols.namesake law medicine TaqMan Humans Liquid biopsy Molecular Biology Polymerase chain reaction 030304 developmental biology Aged Sanger sequencing Aged 80 and over 0303 health sciences Chemistry 010401 analytical chemistry Cancer Cell Biology DNA Neoplasm Middle Aged medicine.disease Molecular biology 0104 chemical sciences Mutation symbols Female KRAS Colorectal Neoplasms |
| Zdroj: | Analytical biochemistry. 590 |
| ISSN: | 1096-0309 |
| Popis: | Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses. The end-point measurement of melt peaks areas by PeakFit software, a nonlinear iterative curve-fitting program, permitted quantification of the mutant/wild-type allele ratios. Approximately 6% and 0.1% of mutant KRAS allele in an excess of wild-type allele is detected with the standard and mePCR-qDMA processes, respectively. The application of the approach was tested for detecting the KRAS codon 12/13 mutation in paired tumor and blood plasma samples from 20 colorectal cancer patients. KRAS mutants were detected in 7 and 18 FFPE tumor samples, and in 3 and 7 plasma samples by the standard and mePCR-qDMA process, respectively. The results were confirmed by Sanger sequencing. This simple, rapid, cost-effective, and quantitative method carried out in a closed-tube format could be applied for the clinical analyses of other cancer genes. |
| Databáze: | OpenAIRE |
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