TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes
| Autor: | Valentina N. Kondratova, Anna Stroganova, Anastasia I. Senderovich, Irina O Panchuk, V. P. Shelepov, I. V. Botezatu, Anatoly V. Lichtenstein |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Proto-Oncogene Proteins B-raf
0301 basic medicine DNA Mutational Analysis Mutant Nucleic Acid Denaturation Polymerase Chain Reaction Sensitivity and Specificity General Biochemistry Genetics and Molecular Biology GTP Phosphohydrolases law.invention 03 medical and health sciences Nucleic acid thermodynamics symbols.namesake 0302 clinical medicine law Primer dimer TaqMan Humans Digital polymerase chain reaction Polymerase chain reaction Sanger sequencing Chemistry Temperature Membrane Proteins DNA Molecular biology 030104 developmental biology 030220 oncology & carcinogenesis symbols Pyrosequencing DNA Probes Biotechnology |
| Zdroj: | BioTechniques. 62:62-68 |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/000114515 |
| Popis: | Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ∼5% and 15%–20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ∼10-fold increase in the detection sensitivity for mutant BRAF and NRAS genes was achieved. |
| Databáze: | OpenAIRE |
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