APPLICATION OF SINGLE-MOLECULE DETECTION TO DNA-SEQUENCING AND SIZING

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per se cond. Our approach relies on fluorescent labeling of the bases in a si ngle fragment of DNA, attachment of this labeled DNA fragment to a sup port, movement of the supported DNA into a flowing sample stream, and detection of the individual, fluorescently labeled bases by laser-indu ced fluorescence as they are cleaved from the DNA fragment by an exonu clease. The ability to sequence large fragments of DNA will reduce sig nificantly the amount of subcloning and the number of overlapping sequ ences required to assemble megabase segments of sequence information. We are also applying our sensitive fluorescence detection to sizing of DNA fragments. Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a f ocused, continuous-wave laser beam in an ultrasensitive flow cytometer at a rate of approximately 60 fragments per second. The size of the f luorescence burst emitted by each stained fragment as it passes throug h the laser beam is measured in one millisecond. We have demonstrated flow cytometric sizing of DNA fragments in a approximately 0.1-pg samp le of a restriction digest of lambda DNA in 164 seconds with sizing ac curacy better than 98%.