RAPID SEPARATION OF DNA-SEQUENCING REACTION-PRODUCT USING CAPILLARY ELECTROPHORESIS

The effect of the gel composition, electric field strength, and capillary length on the separation of DNA sequencing reaction product was investigated in order to achieve high-speed DNA sequencing on a large-scale for the Human Genome Project. In this study, we prepared gel-filled capillaries differing in gel concentration (3% T similar to 6% T), degree of crosslinking (0% C similar to 5% C), and capillary length (15.3 cm similar to 36.3 cm). Fluorescence-labeled A-termination sequencing reaction product (M13mp18 template) was first separated by capillary electrophoresis using the gel-filled capillaries prepared here in electric fields from 200 V/cm to 414 V/cm and then detected at 560 nm by using an on-line argon ion laser (488 nm)-induced fluorescence detector. In conclusion, the use of the short capillary, high concentration gel, and high electric field realizes high-speed separation of DNA sequencing reaction product with high resolution. Under optimum separation conditions, only 10 min is required for sequencing of 250-base DNA.