A 265-Base DNA Sequencing Read by Capillary Electrophoresis with No Separation Matrix

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• 作者：Jennifer Coyne Albrecht ; Jennifer S. Lin ; Annelise E. Barron
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：January 15, 2011
• 年：2011
• 卷：83
• 期：2
• 页码：509-515
• 全文大小：273K
• 年卷期：v.83,no.2(January 15, 2011)
• ISSN：1520-6882

文摘

Electrophoretic DNA sequencing without a polymer matrix is currently possible only with the use of some kind of 鈥渄rag-tag鈥?as a mobility modifier. In free-solution conjugate electrophoresis (FSCE), a drag-tag attached to each DNA fragment breaks linear charge-to-friction scaling, enabling size-based separation in aqueous buffer alone. Here we report a 265-base read for free-solution DNA sequencing by capillary electrophoresis using a random-coil protein drag-tag of unprecedented length and purity. We identified certain methods of protein expression and purification that allow the production of highly monodisperse drag-tags as long as 516 amino acids, which are almost charge neutral (+1 to +6) and yet highly water-soluble. Using a four-color LIF detector, 265 bases could be read in 30 min with a 267-amino acid drag-tag, on par with the average read of current next-gen sequencing systems. New types of multichannel systems that allow much higher throughput electrophoretic sequencing should be much more accessible in the absence of a requirement for viscous separation matrix.