RAPID DNA-SEQUENCING OF MORE THAN 1000 BASES PER RUN BY CAPILLARY ELECTROPHORESIS USING REPLACEABLE LINEAR POLYACRYLAMIDE SOLUTIONS

The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended t o more than 1000 bases with a run time of 80 min. This result was succ essfully achieved through the combined use of cycle sequencing with dy e-labeled primers, improved matrix and separation conditions, and enha nced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the sepa ration buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintai ning an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (>5.5 x 10(6) Da), elevated col umn temperature (50 degrees C) and moderately high field (150 V/cm), r apid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.