Progress towards DNA sequencing at the single molecule level

We describe progress towards sequencing DNA at the single molecule level. Our technique involves incorporation of fluorescently tagged nucleotides into a targeted sequence, anchoring the labeled DNA strand in a flowing stream, sequential exonuclease digestion of the DNA strand, and efficient detection and identification of single tagged nucleotides. Experiments demonstrating strand specific exonuclease digestion of fluorescently labeled DNA anchored in flow as well as the detection of single cleaved fluorescently tagged nucleotides from a small number of anchored DNA fragments axe described. We find that the turnover rate of Esherichia coli exonuclease III on fluorescently labeled DNA in flow at 36{degree}C is {approximately}7 nucleotides per DNA strand per second, which is approximately the same as that measured for this enzyme on native DNA under static, saturated (excess enzyme) conditions. Experiments demonstrating the efficient detection of single fluorescent molecules delivered electrokinetically to a {approximately}3 pL probe volume are also described