DOE Human Genome Program Contractor-Grantee
23. Turn Geometries for Minimizing Band Broadening in Microfabricated Capillary Electrophoresis Channels
Brian M. Paegel, Lester D. Hutt, Peter C. Simpson, and Richard A. Mathies
Department of Chemistry, University of California, Berkeley, CA 94720
Microfabricated capillary electrophoresis (CE) devices have dramatically increased the speed and performance of chemical and biochemical analyses1. As larger numbers of microfabricated structures are placed on a wafer to form capillary array electrophoresis microplates2, or as one attempts to fabricate longer channels to enhance resolution in sequencing applications3, it becomes necessary to fold channels. Folding CE channels gives rise to turn-induced band broadening due to dispersion forces in the turn. To circumvent this limitation, microfabricated channels were constructed with a variety of turn geometries for the purpose of minimizing turn-induced band broadening. Column efficiencies of channels with a variety of turn designs were determined by quantitating the resolution of separations of HaeIII digests of phiX174 bacteriophage DNA. Most advantageously, tapered turns were created by narrowing the channel width at the start of a turn to reduce the channel width, followed by widening the channel at the end of the turn. The radius of curvature of the turn, the length of the tapered region, and the degree of tapering were explored. These experiments were performed using our novel rotary scanner4, which permits the simultaneous interrogation of a separation at three or more points along a serpentine channel. Serpentine channels were monitored before the first turn, after one turn, and after two turns. Turns with a minimum radius of curvature (250 Ám), a minimum length of taper (55 Ám), and a maximum tapering ratio (4:1 separation channel width to turn channel width) were found to provide the highest number of theoretical plates for the 271 and 281 base pair fragments of the phiX174 HaeIII ladder. The optimal turn configuration was then used to perform M13 DNA sequencing separations with an effective separation length of 15 cm. High-quality separations to 800 bp were observed in only 35 minutes. These extended length channel designs have been incorporated in high-throughput 96-channel microplates for DNA sequencing5.
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