Tian H, Brody L C, Mao D, Landers J P
Department of Chemistry, University of Pittsburgh, Pennsylvania 15260, USA.
Anal Chem. 2000 Nov 1;72(21):5483-92. doi: 10.1021/ac0004916.
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, (gamma-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylprrolidone) (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.
通过异源双链分析（HDA）检测DNA突变的毛细管电泳的功效取决于毛细管表面的有效钝化以及用于筛分的正确聚合物网络的选择。使用HDA结合激光诱导荧光检测荧光标记的DNA片段，寻求有效的涂层和最佳的聚合物基质。通过对多种不同的硅烷化试剂、聚合物涂层和聚合物网络进行方法学评估，确定了优化的分离条件，以解析与乳腺癌易感基因（BRCA1）中的五个突变（185delAG、1294del40、4446C>G、5382insC、5677insA）相关的PCR扩增DNA片段。对于毛细管涂层，评估了烯丙基二甲基氯硅烷、4-氯丁基二甲基氯硅烷、（γ-甲基丙烯酰氧基丙基）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烷化试剂，并结合聚乙烯吡咯烷酮（PVP）和聚丙烯酰胺（PA）作为聚合物涂层。将HDA结果与使用商业（FC）涂层毛细管获得的结果进行比较。其中，发现OCT-PVP组合最有效。使用这种改性毛细管，结合包括羟乙基纤维素（HEC）、线性聚丙烯酰胺和PVP的聚合物网络的HDA表明，PVP、PA或FC涂层的毛细管与HEC作为筛分聚合物结合使用，可以在不到10分钟的时间内有效区分突变。然而，使用OCT-PVP涂层毛细管和HEC作为聚合物网络时观察到最佳性能。
