DNA melting analysis: Application of the “open tube” format for detection of mutant KRAS

Scanning of mutations in short amplicons: Optimization of DNA melting method

“…This failure led us to use the analysis of heterodu plexes, which are formed in denaturation-reassocia tion cycles of normal and mutant amplicons, and develop the open tube format, which favors the most effective identification of heteroduplexes [17]. While working in the open tube format, it is possible, first, to maximally decrease the size of the analyzed ampli cons, which increases the probability of identifying the mutation (T melt of DNA decreases on average by 1.0-1.5°C at a decrease in 1% duplex homology [24]) and, second, to optimize the ion content of DNA melting medium.…”

“…The open tube format, which we have suggested previously (post PCR ampl icon shortening and optimization of ion composition of the medium [17]), allows, at the expense of some additional labor, to increase sensitivity of the analysis and makes it suitable for (a standard RT PCR instru ment and widely used SYBR Green 1 dye.…”

“…(b) Low level system (iQ5/standard dye SYBR Green, I/open tube format) [17]. K RAS fragment of 55 bp was amplified by RT PCR on iQ5 (Bio Rad Laboratories) with SYBR Green I (dilution 1 : 75000).…”

“…When necessary, biotinylated KRAS 55 r primer allows one carry out the additional purification of amplicons from disturbing admixtures by immobiliz ing the former on magnetic Streptavidin coupled Dynabeads (Invitrogen Dynal AS, Norway [17]) immediately before the restriction enzyme treatment.…”

Diagnostic significance of DNA and antibodies against capsid antigens of anti-Epstein–Barr virus antibodies levels in blood plasma of nasopharyngeal carcinoma patients from non-endemic region

“…Для ПЦР-РВ применяли праймеры к фрагменту размером 76 пар нуклеотидов в области BamHI-W вирусной ДНК (GenBank accession number V01 555): sense primer -W-44F (5'-CCCAA-CACTCCACCACACC), antisense primer -W-119R (5'-TCTTAGGAGCTGTCCGAGGG), флюоресцент-ный зонд -W-67T (5'-FAMCACACACTACACACACC-CACCCGTCTC-RTQ1) [22]. В качестве контроля спо-собности ДНК плазмы служить матрицей в ПЦР использовали уникальный ген KRAS, как описано ранее [27]. Реакцию вели в 96-луночных планшетах на приборе CFX96 (Bio-Rad Laboratories, США) в 50 мкл реакционной смеси («Синтол», Россия), со-держащей 0,3 мкМ каждого из праймеров, 25 нМ флю-оресцентного зонда, 4 мМ MgCl 2 , 200 мкМ каждого дезоксинуклеозидтрифосфата, 1 ед.…”