Highly sensitive scanning of gene mutations: TaqMan probes as blocking agents
DNA Melting Analysis is very effective in clinical DNA diagnostics: it is simple to perform, high throughput, labor-, time- and cost-effective and is implemented in the “closed tube” format minimizing the risk of samples cross-contamination. Although more sensitive than sequencing by Sanger (mutant...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | Russian |
| Published: |
ABV-press
2016-06-01
|
| Series: | Успехи молекулярной онкологии |
| Subjects: | |
| Online Access: | https://umo.abvpress.ru/jour/article/view/61 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | DNA Melting Analysis is very effective in clinical DNA diagnostics: it is simple to perform, high throughput, labor-, time- and cost-effective and is implemented in the “closed tube” format minimizing the risk of samples cross-contamination. Although more sensitive than sequencing by Sanger (mutant allele detection limit is ~5 and ~15 % respectively), it, however, is inferior in this respect to some other, more laborious and expensive methods (in particular, ddPCR (digital droplet PCR)). Using the BRAF gene as a prototype, we developed the original version of the DNA melting analysis, based on the ability of TaqMan probes to hamper the primer extension reaction by Taq-polymerase. It is found that the weaker blocking effect on the mutant template, which is due to the mismatch in the probe-DNA heteroduplex, permits enriched amplification of the mutant allele and provides a significant (10-fold or more) increase in sensitivity of mutation scanning. |
|---|---|
| ISSN: | 2313-805X 2413-3787 |