Two-color excitation system for fluorescence detection in DNA sequencing by capillary array electrophoresis.
Two computer-controlled galvanometer scanners are adapted for two-dimensional step scanning across a 96-capillary array for laser-induced fluorescence detection. 488 nm and 514 nm laser lines from the same Ar(+) laser were alternately coupled for two-color excitation in each capillary. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries and the excitation wavelengths. Based on the differences in absorption spectra for the dyes, the peak-height ratios in the 488 nm and 514 nm excitation electropherograms were used for peak identification for multiplexed capillary electrophoresis. Successful base calling for 24-capillary DNA sequencing was achieved to 450 bp with 99% accuracy. Advantages include the efficient utilization of light due to the high duty-cycle of step scan, good detection performance due to the reduction of stray light, ruggedness due to the small mass of the galvanometer mirror, low cost due to the simplicity of components and flexibility due to the independent paths for excitation and emission.
Xue
Gang
G
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Yeung
Edward S
ES
D003116:Color; D003365:Costs and Cost Analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D017422:Sequence Analysis, DNA / Q000379:methods*
D005452:Fluoresceins; D005456:Fluorescent Dyes