Abstract

One of the most popular application of the single-molecule detection (SMD) technique is fast DNA sequencing on the single-molecule level as proposed by Keller and coworkers.1,2 The principle idea of this very elegant method involves the incorporation of fluorescently labeled mononucleotides in a growing DNA strand, attachment of a single labeled DNA to a support (generally latex beads), movement of the supported DNA into a flowing sample stream, microchannel or microcapillary3 and detection of the analyte molecules as they are cleaved from the DNA strand by an exonuclease. The DNA sequence is determined by the order in which differently labeled nucleotides are detected and identified. Although a lot of problems are associated with the enzymatic incorporation of labeled mononucleotides and the selective handling of a single DNA strand different detection and identification strategies have been developed. However, up to now, only two different dyes have been successfully identified on the single-molecule level in aqueous solution due to their different fluorescence lifetimes.4,5 Hence, the critical question in DNA sequencing on the single strand is how many labels can be identified on the single-molecule level in aqueous solvent systems.

© 1998 Optical Society of America