Wolfe Kelley A, Breadmore Michael C, Ferrance Jerome P, Power Mary E, Conroy John F, Norris Pamela M, Landers James P
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Electrophoresis. 2002 Mar;23(5):727-33. doi: 10.1002/1522-2683(200203)23:5<727::AID-ELPS727>3.0.CO;2-O.
A silica-based solid-phase extraction system suitable for incorporation into a microchip platform (nu-total analytical system; nu-TAS) would find utility in a variety of genetic analysis protocols, including DNA sequencing. The extraction procedure utilized is based on adsorption of the DNA onto bare silica. The procedure involves three steps: (i) DNA adsorption in the presence of a chaotropic salt, (ii) removal of contaminants with an alcohol/water solution, and (iii) elution of the adsorbed DNA in a small volume of buffer suitable for polymerase chain reaction (PCR) amplification. Multiple approaches for incorporation of this protocol into a microchip were examined with regard to extraction efficiency, reproducibility, stability, and the potential to provide PCR-amplifiable DNA. These included packing microchannels with silica beads only, generating a continuous silica network via sol-gel chemistry, and combinations of these. The optimal approach was found to involve immobilizing silica beads packed into the channel using a sol-gel network. This method allowed for successful extraction and elution of nanogram quantities of DNA in less than 25 min, with the DNA obtained in the elution buffer fraction. Evaluation of the eluted DNA indicated that it was of suitable quality for subsequent amplification by PCR.
一种适用于集成到微芯片平台（全分析系统；μ-TAS）的基于硅胶的固相萃取系统，将在包括DNA测序在内的各种基因分析方案中发挥作用。所采用的萃取程序基于DNA在裸露硅胶上的吸附。该程序包括三个步骤：（i）在离液盐存在下进行DNA吸附，（ii）用醇/水溶液去除污染物，以及（iii）在少量适合聚合酶链反应（PCR）扩增的缓冲液中洗脱吸附的DNA。就萃取效率、重现性、稳定性以及提供可用于PCR扩增的DNA的潜力而言，研究了将该方案集成到微芯片中的多种方法。这些方法包括仅用硅胶珠填充微通道、通过溶胶-凝胶化学生成连续的硅胶网络以及这些方法的组合。发现最佳方法是使用溶胶-凝胶网络固定填充在通道中的硅胶珠。该方法能够在不到25分钟的时间内成功萃取和洗脱纳克量的DNA，洗脱缓冲液部分获得的DNA。对洗脱的DNA的评估表明，其质量适合随后通过PCR进行扩增。
