In this manuscript, we discuss the use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) prior to gel electrophoretic sorting of these DNAs. An immobilization bed for the DNA purification was produced by exposing a posted microchannel to UV radiation, which induced a surface photooxidation reaction, resulting in the production of carboxylate groups. The immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for preconcentration. By suspending the sequencing cocktail in an immobilization buffer (TEG/ethanol), the DNA fragments demonstrated a high affinity for this carboxylated surface. The loading density of DNAs to this activated surface was found to be 3.9 pmol cm(-2). The captured DNA could be subsequently released from the surface by incubation with ddH2O. SPRI cleanup of dye-terminator sequencing fragments using the photoactivated PC chip and slab gel electrophoresis produced a read length comparable to the conventional SPRI format, which utilized carboxylated magnetic beads and a magnetic field. The read length for the PC-SPRI format was found to be 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis (CGE) system. The PC-SPRI method was shown to effectively remove excess dye terminator from the CGE tract, but yielded lower plate numbers, as compared to a direct injection method with purification accomplished off-chip. The loss in efficiency was found to result primarily from the extended injection time associated with the microchip purification method.

中文翻译：

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固相可逆固定在微流控芯片中，用于纯化染料标记的DNA测序片段。

在此手稿中，我们讨论了在将这些DNA进行凝胶电泳分选之前，使用固相可逆固定化（SPRI）使用光活化聚碳酸酯（PC）纯化染料标记的终止子测序片段。通过将张贴的微通道暴露于紫外线辐射中来生产用于DNA纯化的固定床，这会引发表面光氧化反应，从而产生羧酸酯基团。固定微通道包含微柱，可增加DNA的负载水平，从而改善信号强度，而无需预先浓缩。通过将测序混合物悬浮在固定化缓冲液（TEG /乙醇）中，DNA片段显示出对该羧基化表面的高度亲和力。发现DNA对该活化表面的装载密度为3.9 pmol cm（-2）。随后可以通过与ddH2O孵育从表面释放捕获的DNA。使用光活化的PC芯片和平板凝胶电泳对染料终止剂测序片段进行SPRI纯化，产生的读取长度可与传统SPRI格式相媲美，传统SPRI格式利用了羧基化的磁珠和磁场。发现PC-SPRI格式的读取长度为620个碱基，呼叫精度为98.9％。PC-SPRI清除格式也已集成到毛细管凝胶电泳（CGE）系统中。与直接进样且芯片外完成纯化的方法相比，PC-SPRI方法已被证明可以有效地从CGE通道中去除过量的染料终止剂，但产生的塔板数更少。