Highly sensitive scanning of gene mutations: TaqMan probes as blocking agents
https://doi.org/10.17650/2313-805X.2016.3.2.42-49
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.
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
Russian Federation
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
Russian Federation
Russian Federation
A. M. Stroganova
Research Institute of Clinical Oncology, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 Kashirskoe
Shosse, Moscow, 115478, Russia
Russian Federation
Russian Federation
A. I. Senderovich
Research Institute of Clinical Oncology, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia; 24 Kashirskoe
Shosse, Moscow, 115478, Russia
Russian Federation
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
Russian Federation
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
Russian Federation
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
Russian Federation
Russian Federation
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Review
For citations:
Botezatu I.V., Panchuk I.O., Stroganova A.M., Senderovich A.I., Kondratova V.N., Shelepov V.P., Liсhtenshtein A.V. Highly sensitive scanning of gene mutations: TaqMan probes as blocking agents. Advances in Molecular Oncology. 2016;3(2):42-49. (In Russ.) https://doi.org/10.17650/2313-805X.2016.3.2.42-49
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