Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing
Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing
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DOI：
10.1021/ac0203645
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发表时间：
2002-10-01
影响因子：
7.4
通讯作者：
Mathies, RA
中科院分区：
文献类型：
--
作者：
Paegel, BM;Yeung, SHI;Mathies, RA
本文介绍了一种集成DNA测序样品分离、模板去除、预浓缩和毛细管电泳分析的微型电泳生物处理器。将含有与M13-40通用正向引发位点的直接3'的20个碱基序列互补的丙烯酰胺共聚寡核苷酸的低粘度凝胶捕获基质引入60-nL捕获室中。未纯化的DNA测序反应产物被电泳驱动通过腔室;延伸产物与基质杂交，而污染的缓冲离子、Cl-、过量的引物和模板DNA未被保留。在优化的条件下，纯化仅在120 s内完成（结合温度50 ℃，驱动电压250 V）。通过在67 ℃下释放凝胶纯化的双链体并直接注射到15.9 cm有效长度的CE微通道上来完成高速、整合的测序分析。测序产物的电泳解析在32分钟内完成，产生总共560 bp，phred质量q大于或等于20（准确度大于或等于99%）。这种完全集成的纳升工艺将纯化时间缩短了近10倍，工艺体积减少了近100倍，同时提供了最先进的测序结果。
A microfabricated electrophoretic bioprocessor for integrated DNA sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide-copolymerized oligonucleotide complementary to the 20-base sequence directly 3' of the M13-40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50degreesC, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gel-purified duplex at 67degreesC and directly injecting onto a 15.9-cm effective length CE microchannel. Electrophoretic resolution of the sequencing products is complete in 32 min, producing a total of 560 bp with phred quality qgreater than or equal to20 (accuracy greater than or equal to99%). This fully integrated nanoliter process decreases the purification time similar to10-fold and the process volume similar to100-fold while providing state-of-the-art sequencing results.