DNA sequencing by capillary electrophoresis: use of a two-laser-two-window intensified diode array detection system

This paper presents the principles of an instrument designed for DNA sequencing using the standard four-dye-labeled primer approach. The method is based on capillary electrophoresis with laser-induced fluorescence and an intensified diode array detector. An important goal of the instrument design has been a detection system that possesses high sensitivity and high spectral resolution. Based on an analysis of the spectral characteristics of the four standard dye-labeled primers, FAM, JOE, 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, followed by a 543-nm helium-neon laser illuminating the second window. The two-window approach has no moving parts and permits continuous illumination. Spectral resolution is provided by a grating spectrograph and a cooled intensified diode array. The estimated limit of detection for the standard four dye-labeled primers was found to be in the sample concentration range of 1 x 10(-12) M. To achieve these low levels, complete free-radical polymerization of polyacrylamide has been found to be necessary in order to reduce background noise. In addition, reduction in background noise was accomplished by continual purging of the anodic reservoir in order to prevent electrolysis products from entering the capillary. Separation of DNA sequencing reaction products is demonstrated on a 9% T linear polyacrylamide column.