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

Abstract

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.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

“…To compensate for this effect, one must use more template DNA in the reactions involving rhodamine dye-labeled primers. One way to solve this problem is to use multiple lasers (10,11). A The publication costs of this article were defrayed in part by page charge payment.…”

Color-blind fluorescence detection for four-color DNA sequencing

“…Other multiple laser systems have been reported for DNA sequencing applications, which resolve fluorescent light into color for base-calling purposes (17,(31)(32)(33). In a recent report, Alaverdian et al (19) also used four lasers, which were all operated in a continuous-wave mode but modulated at different RFs.…”

DNA Sequencing by Capillary Electrophoresis Using Short Oligonucleotide Primer Libraries

“…The instrument described in this paper differs only in the detector and the capillary column heating device. The detector is an intensified photodiode array instrument (Model 1420; EG&G Princeton Applied Research, Princeton, NJ, USA) for full spectral detection of labeled dideoxynucleotides (5). The thermostat on the heating device regulates the temperature of the separation column, providing constant temperature (e.g., 30° ± 0.5°C) for 15 cm of the effective length of the capillary.…”

“…The schematic of the data-acquisition system has been published previously (5). Stored data are analyzed with the aid of the laboratory-developed software, which allows viewing of the four-color dye channels, spectral information, base calling, etc.…”