Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis

Abstract

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.

Capillary electrophoresis of catecholamines with laser‐induced fluorescence intensified charge‐coupled device detection

“…Sweedler et al (1991) utilized the time-delayed integration mode to detect subattomole quantity of fluorescein isothiocyanate-labeled amino acids and neuropeptides. The multi-wavelength fluorescence electropherograms detected by CCD were used for rapid DNA sequencing (Karger et al, 1991), purity analysis of fluorescence agents (Xiong et al, 1996b), determination of tryptophan and tyrosine contents of peptides (Timperman et al, 1995), and multicomponent analysis of highly overlapped capillary electrophoretic peaks (Xiong et al, 1996c). This laboratory has established a capillary electrophoresis-laser-induced fluorescence-intensified charge-coupled device (CE-LIF-ICCD) system.…”

Characteristics of single‐stranded DNA separation by capillary gel electrophoresis

“…Typically, the capillaries for the DNA sequencing are filled with cross-linked polyacrylamide gel [2,4,7,11,121 or noncross-linked polyacrylamide (Le,, linear polyacrylamide [5, 7, 10, 13, 171. More recently, DNA sequencing by CGE with linear polyacrylamide is being increasingly studied [13, 171 and linear polyacrylamide is becoming an important molecular sieving matrix because linear polyacrylamide can be replaced by fresh medium after the separation.…”

“…Capillaries filled with cross-linked polyacrylamide gel [2,4,7,11,121 or noncross-linked polyacrylamide [5,7,10,13,171 are widely used for the DNA sequencing by CGE. We used a 4%T, 5%c cross-linked polyacrylamide gel as separation matrix.…”

Capillary electrophoresis with wavelength-resolved laser-induced fluorescence detection

“…LOD were improved dramatically to 20-80 zmole (13 nL injection) for fluorescein isothiocyanate-labeled amino acids. Also in 1991 a CCD-LIF system with a 94% duty cycle, which furnished high-quality spectral information, was demonstrated [55]. The concentration LOD for Joe-A oligonucleotide primer was 26 pmol L -1 .…”