Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels.
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.
Liu
S
S
Department of Chemistry, University of California, Berkeley 94720, USA.
Shi
Y
Y
Ja
W W
WW
Mathies
R A
RA
D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*