Advances in Molecular OncologyAdvances in Molecular OncologyУспехи молекулярной онкологии2313-805X2413-3787Publishing House ABV Press6110.17650/2313-805X.2016.3.2.42-49RESEARCH ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИHighly sensitive scanning of gene mutations: TaqMan probes as blocking agentsВысокочувствительное сканирование генных мутаций: зонды TaqMan как блокирующие агентыBotezatuI. V.БотезатуИ. В.
Russian Federation
PanchukI. O.ПанчукИ. О.
Russian Federation
StroganovaA. M.СтрогановаА. М.
Russian Federation
SenderovichA. I.СендеровичА. И.
Russian Federation
KondratovaV. N.КондратоваВ. Н.
Russian Federation
ShelepovV. P.ШелеповВ. П.
Russian Federation
LiсhtenshteinA. V.ЛихтенштейнА. В.
Russian Federation
alicht@mail.ruResearch Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of RussiaНИИ канцерогенеза ФГБУ «РОНЦ им. Н. Н. Блохина» Минздрава России24 Kashirskoe Shosse, Moscow, 115478, RussiaРоссия, 115478, Москва, Каширское шоссе, 24Research Institute of Clinical Oncology, N. N. Blokhin Russian Cancer Research Center, Ministry of Health of RussiaНИИ клинической онкологии ФГБУ «РОНЦ им. Н. Н. Блохина» Минздрава России24 Kashirskoe Shosse, Moscow, 115478, RussiaРоссия, 115478, Москва, Каширское шоссе, 24150520163242490706201607062016Copyright ©; 2016, Botezatu I.V., Panchuk I.O., Stroganova A.M., Senderovich A.I., Kondratova V.N., Shelepov V.P., Liсhtenshtein A.V.Copyright ©; 2016, Ботезату И.В., Панчук И.О., Строганова А.М., Сендерович А.И., Кондратова В.Н., Шелепов В.П., Лихтенштейн А.В.2016Botezatu I.V., Panchuk I.O., Stroganova A.M., Senderovich A.I., Kondratova V.N., Shelepov V.P., Liсhtenshtein A.V.Ботезату И.В., Панчук И.О., Строганова А.М., Сендерович А.И., Кондратова В.Н., Шелепов В.П., Лихтенштейн А.В.https://creativecommons.org/licenses/by/4.0https://umo.abvpress.ru/jour/article/view/61

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.

Метод плавления ДНК весьма эффективен в клинической генодиагностике, прост в исполнении, производителен, экономичен и, кроме того, реализуется в «закрытом формате», сводящем к минимуму затраты времени, труда и риск перекрестного загрязнения образцов. Данный метод более чувствительный, чем секвенирование по Сэнгеру (предел обнаружения мутантных аллелей ~5 и ~15 % соответственно), однако уступает в этом отношении другим, более трудоемким и дорогим методам (в частности, капельной цифровой полимеразной цепной реакции (digital droplet PCR)). На гене BRAF (как прототипе) мы разработали оригинальный вариант метода плавления ДНК, основанный на способности зондов TaqMan затруднять движение Taq-полимеразы по матрице. Установлено, что эффект блокирования слабее выражен на мутантной матрице из-за присутствия в дуплексе зонд-ДНК неспаренного основания. Предложен протокол полимеразной цепной реакции, дискриминирующий амплификацию мутантных и нормальных аллелей и обеспечивающий существенное (10-кратное и более) повышение чувствительности мутационного сканирования.

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