High-Speed DNA Sequencing by Using Mixed Poly(ethylene oxide) Solutions in Uncoated Capillary Columns
High-Speed DNA Sequencing by Using Mixed Poly(ethylene oxide) Solutions in Uncoated Capillary Columns
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DOI：
10.1021/ac00109a002
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发表时间：
1995-07
影响因子：
7.4
通讯作者：
E. Fung;E. Yeung
中科院分区：
文献类型：
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作者：
E. Fung;E. Yeung
我们描述了一种新的非交联聚合物基质和一种新的柱处理程序，用于通过毛细管电泳进行高速 DNA 测序。两种尺寸范围的市售聚环氧乙烷 (PEO) 溶液用作变性条件下的筛分基质。与市售的 6% T 聚丙烯酰胺溶液相比，混合 PEO 基质对小 DNA 片段具有相同的分离性能，对大 DNA 片段具有更好的性能，提高了分离速度，并降低了粘度。为了避免毛细管柱上的标准粘合涂层在多次运行中降解，我们评估了裸熔融石英毛细管中混合 PEO 基质中 DNA 片段的分辨率。在干燥、新鲜的色谱柱中，其性能与在涂层（键合）毛细管中观察到的性能相同。为了在每次运行后恢复良好的表面特性，使用 0.1 N HCl 将表面硅烷醇基团重新滴定回完全质子化的形式。事实上，测序运行的速度要快得多，Sanger 阶梯中的碱基 28-420 仅在 16 分钟内就被洗脱出来。我们观察到在数十次测序运行中性能几乎没有下降。 36 个参考文献，3 个图。
We describe a new non-cross-linked polymer matrix and a new column treatment procedure for high-speed DNA sequencing by using capillary electrophoresis. A solution of commercially available poly(ethylene oxide) (PEO) in two size ranges is used as the sieving matrix under denaturing conditions. When compared to a commercial 6% T polyacrylamide solution, the mixed PEO matrix offers identical separation performance for the small DNA fragments, better performance for the large DNA fragments, increased separation speed, and a lower viscosity. To avoid degradation of the standard bonded coating on the capillary column over many runs, we evaluated the resolution of DNA fragments in the mixed PEO matrix in bare fused-silica capillaries. In a dry, fresh column, the performance is identical to that observed in coated (bonded) capillaries. To regenerate the favorable surface characteristics after each run, 0.1 N HCl was used to retitrate the surface silanol groups back to the fully protonated form. The sequencing runs in fact proceeded much faster, with bases 28-420 from the Sanger ladder eluting in a span of only 16 min. We observed little degradation of the performance over tens of sequencing runs. 36 refs., 3 figs.