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用于DNA测序的固相支持物样品加载

Solid-support sample loading for DNA sequencing.

作者信息

Ueberfeld Jörn, El-Difrawy Sameh A, Ramdhanie Korisha, Ehrlich Daniel J

机构信息

Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, USA.

出版信息

Anal Chem. 2006 Jun 1;78(11):3632-7. doi: 10.1021/ac052201x.

Abstract

We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel. This method circumvents both the minimum volume requirement of pipettors (since only solid beads are transported) and the timing complications of double-T microfluidic injection. Injections from Sanger samples of <100 pg total suspended weight match the signal strength of our previous conventional injections at >10-times the starting DNA sample. Sequencing traces show a resolution that matches or exceeds double-T injections. A kinetic model reproduces the time-dependence of the injection signals and proves that total nonidealities in the method produce injection-broadened plugs of approximately 1-s duration. The method should be broadly extendable to DNA and protein separations in both microdevice and capillary electrophoresis.

摘要

我们提出了一种将低量DNA简化加载到微电泳装置上的新方法。该方法基于对可逆结合在顺磁性微球上的DNA进行固相萃取、纯化和转运的组合。DNA吸附到微球上,用磁化坡莫合金丝捕获,然后作为高度聚焦的样品塞直接注入分离通道。此方法既规避了移液器的最小体积要求(因为仅运输固体珠),又避免了双T微流体注射的时间复杂性。从总悬浮重量小于100 pg的桑格样品进行的注射,其信号强度与我们之前使用起始DNA样品10倍以上的传统注射信号强度相当。测序图谱显示的分辨率与双T注射相当或更高。动力学模型再现了注射信号的时间依赖性,并证明该方法中的总非理想性会产生持续时间约为1秒的注射展宽塞。该方法应可广泛扩展到微装置电泳和毛细管电泳中的DNA和蛋白质分离。

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