Woolley A T, Mathies R A
Department of Chemistry, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11348-52. doi: 10.1073/pnas.91.24.11348.
Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of phi X174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 microns) have been explored. By use of channels with an effective length of only 3.5 cm, separations of phi X174 Hae II DNA fragments from approximately 70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQ alpha alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips.
通过光刻掩膜和化学蚀刻技术，在平面玻璃基板上制作了毛细管电泳阵列。在玻璃基板上蚀刻光刻定义的通道图案，然后通过将蚀刻后的基板热键合到另一块载玻片上形成毛细管。使用通道中的羟乙基纤维素筛分基质，对这些芯片进行了phi X174 Hae III DNA限制性片段的高分辨率电泳分离。DNA片段在运行缓冲液中用染料进行荧光标记，并用激光激发的共聚焦荧光系统进行检测。研究了电场变化、进样程序以及分离和进样通道尺寸（范围为30至120微米）的影响。通过使用有效长度仅为3.5厘米的通道，仅在120秒内就完成了phi X174 Hae II DNA片段从约70至1000 bp的分离。我们还展示了PCR扩增的HLA-DQα等位基因的高速大小测定。这项工作建立了在毛细管阵列电泳芯片上进行高速、高通量DNA分离的方法。
