TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes
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 a...
| Veröffentlicht in: | BioTechniques. - 1988. - 62(2017), 2 vom: 01. Feb., Seite 62-68 |
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| Weitere Verfasser: | , , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2017
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't BRAF DNA melting analysis NRAS TaqMan probes blocking agents mutation detection DNA Probes Membrane Proteins mehr... |
| Zusammenfassung: | 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 |
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| Beschreibung: | Date Completed 25.10.2017 Date Revised 06.03.2018 published: Electronic Citation Status MEDLINE |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/000114515 |