Effective capillary electrophoresis-based heteroduplex analysis through optimization of surface coating and polymer networks.
Effective capillary electrophoresis-based heteroduplex analysis through optimization of surface coating and polymer networks.
复制标题
通过优化表面涂层和聚合物网络，进行有效的基于毛细管电泳的异源双链分析。
DOI：
10.1021/ac0004916
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发表时间：
2000
影响因子：
7.4
通讯作者：
Landers,JP
中科院分区：
文献类型：
--
作者：
Tian,H;Brody,LC;Mao,D;Landers,JP
毛细管电泳通过异质双工分析（HDA）检测DNA突变的有效性取决于毛细管表面的有效钝化和选择正确的聚合物网络进行筛分。利用HDA激光诱导荧光检测荧光标记的DNA片段，寻找有效的涂层和最佳聚合物基质。通过对不同硅化试剂、聚合物涂层和聚合物网络的方方学评价，确定了分离乳腺癌易感基因BRCA1中5个突变（185delAG、1294del40、4446C>G、532insc、5677insA）相关pcr扩增DNA片段的最佳分离条件。在毛细管涂层中，以聚乙烯基吡咯烷酮（PVP）和聚丙烯酰胺（PA）为聚合物涂层，对烯丙基二甲基氯硅烷、4-氯丁基二甲基氯硅烷、（γ-甲基丙烯氧基）三甲氧基硅烷、氯二甲基辛基硅烷（OCT）和7-辛烯基三甲氧基硅烷作为硅化剂进行了评价。HDA结果与商用（FC）涂膜毛细管的结果进行了比较。其中，OCT - PVP组合是最有效的。利用这种改进的毛细管，HDA与包括羟乙基纤维素（HEC），线性聚丙烯酰胺和PVP在内的聚合物网络表明，PVP-， PA-或fc包被的毛细管，结合HEC作为筛选聚合物，可以在不到10分钟的时间内有效地识别突变。然而，以OCT−pvp包被的毛细管和HEC作为聚合物网络，观察到最佳的性能。
The efficacy of capillary electrophoresis for detecting DNA mutations via heteroduplex analysis (HDA) is dependent upon both the effective passivition of the capillary surface and the choice of the correct polymer network for sieving. Using HDA with laser-induced fluorescence detection of fluorescently labeled DNA fragments, an effective coating and optimal polymer matrix were sought. Optimized separation conditions were determined through the methodological evaluation of a number of different silanizing reagents, polymeric coatings, and polymer networks for resolving the PCR-amplified DNA fragments associated with five mutations (185delAG, 1294del40, 4446C>G, 5382insC, 5677insA) in the breast cancer susceptibility gene (BRCA1). For capillary coating, allyldimethylchlorosilane, 4-chlorobutyldimethylchlorosilane, (γ-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylpyrrolidone) (PVP) and polyacrylamide (PA) as the polymeric coat. The HDA results were compared with those obtained using a commercial (FC) coated capillary. Of these, the OCT−PVP combination was found to be most effective. Using this modified capillary, HDA with polymer networks that included hydroxyethylcellulose (HEC), linear polyacrylamide, and PVP showed that a PVP-, PA-, or FC-coated capillary, in combination with HEC as the sieving polymer, could be used effectively to discriminate the mutations in less than 10 min. However, optimal performance was observed with the OCT−PVP-coated capillary and HEC as the polymer network.