DNA-SEQUENCING BY CAPILLARY ELECTROPHORESIS - USE OF A 2-LASER 2-WINDOW INTENSIFIED DIODE-ARRAY DETECTION SYSTEM

This paper presents the principles of an instrument designed for DNA s equencing using the standard four-dye-labeled primer approach. The met hod is based on capillary electrophoresis with laser-induced fluoresce nce and an intensified diode array detector. An important goal of the instrument design has been a detection system that possesses high sens itivity and high spectral resolution. Based on an analysis of the spec tral characteristics of the four standard dye-labeled primers, FAM, JO E, ROX, and TAMRA, the strategy has been to use a two-laser-two-window approach, in which a 488-nm argon ion laser illuminates one window, f ollowed by a 543-nm helium-neon laser illuminating the second window. The two-window approach has no moving parts and permits continuous ill umination. Spectral resolution is provided by a grating spectrograph a nd a cooled intensified diode array. The estimated limit of detection for the standard four dye-labeled primers was found to be in the sampl e concentration range of 1 x 10(-12) M. To achieve these low levels, c omplete free-radical polymerization of polyacrylamide has been found t o be necessary in order to reduce background noise. In addition, reduc tion in background noise was accomplished by continual purging of the anodic reservoir in order to prevent electrolysis products from enteri ng the capillary. Separation of DNA sequencing reaction products is de monstrated on a 9% T linear polyacrylamide column.