Integrated electroosmotically-driven on-line sample purification system for nanoliter DNA sequencing by capillary electrophoresis

Abstract

An integrated on-line system is developed for DNA sequencing at the nanoliter scale. The technique involves the use of a nanoreactor for small-volume cycle-sequencing reaction, capillary zone electrophoresis (CZE) for purification of the sequencing fragments, and capillary gel electrophoresis (CGE) for separation of the purified DNA fragments. The nanoreactor and CZE are integrated into one capillary, where a 100-nl dye-labeled terminator cycle-sequencing reaction is carried out followed by CZE to separate excess dye-labeled terminators from the sequencing fragments. On-line electrokinetic injection of the purified DNA fragments into the CGE system is accomplished at a small-volume tee connector by which the CZE capillary is interfaced to the CGE system. The utility of the system is demonstrated in sequencing nanoliter volumes of single-stranded DNA (M13mp18) and double-stranded DNA (pGEM). The use of voltage to drive both CZE and CGE makes it feasible for automation and future adaptation of the whole system to a microchip.