DNA sequencing by capillary electrophoresis with four-decay fluorescence detection

A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequen cing is described in which on-the fly, frequency-domain fluorescence lifeti me 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 w ere evaluated, one excited at 488 nm and the other, at 514 nm. The 488 nm s ystem proved successful for four-decay detection. Base calling was achieved either directly from on-the-fly lifetimes or from lifetime-resolved electr opherograms recovered for each base from the electropherogram of the mixtur e of sequencing reaction products. The latter method was found to be more a ccurate (99% for two bases and 98.5% for three bases) and could achieve lon ger 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 sequenc ing over the fragment length range of 41220 bases.