Department of Chemistry, Louisiana State University, Baton Rouge, LA, USA.
Electrophoresis. 2011 Nov;32(22):3221-32. doi: 10.1002/elps.201100274. Epub 2011 Oct 31.
An integrated and modular DNA analysis system is reported that consists of two modules: (i) A continuous flow polymerase chain reaction (CFPCR) module fabricated in a high T(g) (150°C) polycarbonate substrate in which selected gene fragments were amplified using biotin and fluorescently labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones as opposed to heating and cooling large thermal masses typically performed in batch-type thermal reactors. (ii) μCE (micro-capillary electrophoresis) module fabricated in poly(methylmethacrylate) (PMMA), which utilized a bioaffinity selection and purification bed (2.9 μL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to electrophoretic sorting. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed, where they were extracted onto the surface of micropillars. The affinity bed was also fabricated in PMMA and was populated with an array of microposts (50 μm width; 100 μm height) yielding a total surface area of ∼117 mm(2). This solid-phase extraction (SPE) process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (K(d) = 10(-15) M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single-stranded DNA released for injection into a 7-cm-long μCE channel for size-based separations and fluorescence detection. The utility of the system was demonstrated using Alu DNA typing for gender and ethnicity determinations as a model. Compared with the traditional cross-T injection procedure typically used for μCE, the affinity pre-concentration and injection procedure generated signal enhancements of 17- to 40-fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation.
本文报道了一种集成式和模块化 DNA 分析系统，它由两个模块组成：(i) 在高玻璃化转变温度（150°C）聚碳酸酯基板上制造的连续流聚合酶链反应（CFPCR）模块，其中使用生物素和荧光标记的引物对选定的基因片段进行扩增，通过连续穿梭小体积的 PCR 试剂和模板穿过等温区来实现，而不是像批量式热反应器那样加热和冷却大的热质量。(ii) 在聚甲基丙烯酸甲酯（PMMA）中制造的微毛细管电泳（μCE）模块，该模块利用生物亲和性选择和纯化床（2.9 μL）在电泳分选之前预浓缩和纯化从 CFPCR 模块生成的 PCR 产物。生物素标记的 CFPCR 产物通过静水压泵入链霉亲和素修饰床，在那里它们被提取到微柱的表面上。亲和床也在 PMMA 中制造，填充有微柱阵列（50 μm 宽；100 μm 高），总表面积约为 117 mm²。这种固相萃取（SPE）过程对生物素化的扩增子表现出高选择性，并利用强链霉亲和素/生物素相互作用（Kd = 10⁻¹⁵ M）实现高回收率。SPE 选择的 CFPCR 产物进行热变性，单链 DNA 释放，用于注入 7 cm 长的 μCE 通道进行基于大小的分离和荧光检测。该系统的实用性通过使用 Alu DNA 分型作为模型来进行性别和种族鉴定来证明。与传统的用于 μCE 的交叉 T 注入程序相比，亲和预浓缩和注入程序产生了 17 到 40 倍的信号增强，这对于 CFPCR 热循环器至关重要，因为它们的操作与泰勒分散有关。
