Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids.
Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids.
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DOI：
10.1002/chem.201101987
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发表时间：
2011-11-11
影响因子：
4.3
通讯作者：
Kolpashchikov, Dmitry M.
中科院分区：
文献类型：
--
作者：
Nguyen, Camha;Grimes, Jeffrey;Gerasimova, Yulia V.;Kolpashchikov, Dmitry M.
Hybridization probes are often inefficient in the analysis of single-stranded DNA or RNA that are folded in stable secondary structures. An MB probe is a short DNA hairpin with a fluorophore and a quencher attached to the opposite sides of the oligonucleotide. The probe is widely used in real-time analysis of specific DNA and RNA sequences. This study demonstrates how conventional molecular beacon (MB) probe can be used for the analysis of nucleic acids that form very stable (Tm >80°C) hairpin structures. Here we demonstrate that MB probe is not efficient in direct analysis of secondary structure-folded analytes, while MB-based tricomponent probe is suitable for these purposes. Tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to an MB probe. In the presence of a specific analyte the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11 or 13 base-pairs can be detected in real time with the limit of detection (LOD) lying in nanomolar range. The stability of the stem region in DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such application as in vivo RNA monitoring, detection of pathogens and SNPs genotyping by DNA microarrays.
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影响因子：
16.6
作者：
Grimes, Jeffrey;Gerasimova, Yulia V.;Kolpashchikov, Dmitry M.
通讯作者：
Kolpashchikov, Dmitry M.
影响因子：
4.4
作者：
Kubota, Kengo;Ohashi, Akiyoshi;Harada, Hideki
通讯作者：
Harada, Hideki
DOI：
10.1039/c0cc03248d
发表时间：
2010-12-14
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Gerasimova YV;Peck S;Kolpashchikov DM
通讯作者：
Kolpashchikov DM
影响因子：
3.2
作者：
Gerasimova, Yulia V.;Cornett, Evan;Kolpashchikov, Dmitry M.
通讯作者：
Kolpashchikov, Dmitry M.
影响因子：
14.9
作者：
Gao Y;Wolf LK;Georgiadis RM
通讯作者：
Georgiadis RM