Optimization of High-Speed DNA Sequencing on Microfabricated Capillary Electrophoresis Channels

Abstract

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.

Peer Reviewed: Polymeric Microelectromechanical Systems.

“…For microseparation-based assays, deep channels mean that more sample can be loaded, which improves detection. Narrow channels mean that the number of turns can be increased without producing significant amounts of zonal dispersion (20,21).…”

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

“…In subsequent work, the classical variables of DNA sequencing separations such as column length, sieving matrix composition, injection parameters, etc. have been explored and optimized (13,14), producing read lengths as long as 800 bp for a 40-cm-long microfabricated capillary in 80 min (15).Microfabricated capillary array electrophoresis ( CAE) devices are advantageous compared with drawn capillary array instrumentation because they allow monolithic array construction, precise definition of picoliter sample injection plugs, and the potential for integrated, low volume sample processing. Our group has pioneered the design and construction of microfabricated capillary array electrophoresis devices demonstrating rectilinear networks of 12 and 48 channels for parallel DNA fragment analysis (16, 17).…”

“…Our group has pioneered the design and construction of microfabricated capillary array electrophoresis devices demonstrating rectilinear networks of 12 and 48 channels for parallel DNA fragment analysis (16, 17). The field was further advanced with the presentation of a 96-lane radial microchannel plate (MCP) layout together with a rotary confocal scanning detector and its use for high throughput genotyping (18).The primary challenge in adapting the radial 96-lane MCP for DNA sequencing is attaining the necessary column lengths (13,14). Folding channels to increase the effective separation length maintains a compact design compatible with modern wafer-scale fabrication.…”

“…In subsequent work, the classical variables of DNA sequencing separations such as column length, sieving matrix composition, injection parameters, etc. have been explored and optimized (13,14), producing read lengths as long as 800 bp for a 40-cm-long microfabricated capillary in 80 min (15).…”

“…The primary challenge in adapting the radial 96-lane MCP for DNA sequencing is attaining the necessary column lengths (13,14). Folding channels to increase the effective separation length maintains a compact design compatible with modern wafer-scale fabrication.…”