Email this article Highly sensitive scanning of gene mutations: TaqMan probes as blocking agents
- Authors: Botezatu I.V.1,2, Panchuk I.O.1,2, Stroganova A.M.3,4, Senderovich A.I.3,4, Kondratova V.N.1,2, Shelepov V.P.1,2, Liсhtenshtein A.V.1,2
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Affiliations:
- Research Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia
- 24 Kashirskoe Shosse, Moscow, 115478, Russia
- Research Institute of Clinical Oncology, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia
- 24 Kashirskoe Shosse, Moscow, 115478, Russia
- Issue: Vol 3, No 2 (2016)
- Pages: 42-49
- Section: RESEARCH ARTICLES
- Published: 07.06.2016
- URL: https://umo.abvpress.ru/jour/article/view/61
- DOI: https://doi.org/10.17650/2313-805X.2016.3.2.42-49
- ID: 61
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Abstract
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.
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About the authors
I. V. Botezatu
Research Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 Kashirskoe Shosse, Moscow, 115478, Russia
Email: fake@neicon.ru
Russian Federation
I. O. Panchuk
Research Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 Kashirskoe Shosse, Moscow, 115478, Russia
Email: fake@neicon.ru
Russian Federation
A. M. Stroganova
Research Institute of Clinical Oncology, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 KashirskoeShosse, Moscow, 115478, Russia
Email: fake@neicon.ru
Russian Federation
A. I. Senderovich
Research Institute of Clinical Oncology, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 KashirskoeShosse, Moscow, 115478, Russia
Email: fake@neicon.ru
Russian Federation
V. N. Kondratova
Research Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 Kashirskoe Shosse, Moscow, 115478, Russia
Email: fake@neicon.ru
Russian Federation
V. P. Shelepov
Research Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 Kashirskoe Shosse, Moscow, 115478, Russia
Email: fake@neicon.ru
Russian Federation
A. V. Liсhtenshtein
Research Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 Kashirskoe Shosse, Moscow, 115478, Russia
Author for correspondence.
Email: alicht@mail.ru
Russian Federation
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