Application of Single Molecule Detection to DNA Sequencing and Sizing

<jats:title>Abstract</jats:title><jats:p>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 second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, and detection of the individual, fluorescently labeled bases by laser‐induced fluorescence as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will reduce significantly the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.</jats:p><jats:p>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 focused, continuous‐wave laser beam in an ultrasensitive flow cytometer at a rate of ∼60 fragments per second. The size of the fluorescence burst emitted by each stained fragment as it passes through the laser beam is measured in one millisecond. We have demonstrated flow cytometric sizing of DNA fragments in a ∼0.1‐pg sample of a restriction digest of lambda DNA in 164 seconds with sizing accuracy better than 98%.</jats:p>