DNA sequencing by capillary electrophoresis with four-decay fluorescence detection.
A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequencing is described in which on-the-fly, frequency-domain fluorescence lifetime detection is used to discriminate among the dye-labeled fragments of the four terminal bases in a single-lane CE separation. Two four-dye systems were evaluated, one excited at 488 nm and the other, at 514 nm. The 488 nm system proved successful for four-decay detection. Base calling was achieved either directly from on-the-fly lifetimes or from lifetime-resolved electropherograms recovered for each base from the electropherogram of the mixture of sequencing reaction products. The latter method was found to be more accurate (99% for two bases and 98.5% for three bases) and could achieve longer read lengths, but it was unsuccessful for sequencing of all four bases. The first method gave a base-calling accuracy of 96% for four-base sequencing over the fragment length range of 41-220 bases.
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Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
McGown
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D001483:Base Sequence; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D017931:DNA Primers