The First Single-Molecule, Single-Base-Resolution DNA Sequencing

The first single-molecule, single-base-resolution DNA sequencing has been carried out by a Caltech group. In this new approach, the bases forming the backbone of the typical DNA molecule are viewed one by one in the act of replicating. To be more exact, a DNA polymerase molecule, acting as a genetic xerox machine, copies a single strand of DNA by adding complementary base units to it; the "fuel" for this process, the base molecules being added, were fluorescently labeled beforehand (by attaching site-specific, light-producing fluorophore molecules), so the DNA sequence could be observed by microscope observations (schematic setup figure). Sequencing single-molecule DNA strands is intrinsically difficult because of the high linear data storage density: the bases are only about 3.4 angstroms apart along the DNA helix. Past efforts to sequence bases through their fluorescence have been complicated by background noise, a problem avoided by the Caltech scientists through careful use of two laser pulses, one for producing pinpoint fluorescence and another for nulling or "bleaching" the fluorescence in order to prepare for the next base identification.

Stephen Quake and his colleagues can currently identify no more than about 6 bases in a row, so this research is still at the proof-of-principle stage. However, within about two years or so, Quake believes, his process should be a factor of ten faster than standard gel-electrophoresis techniques used to sequence DNA molecules on a wholesale level, and several orders of magnitude cheaper. (Braslavsky et al., Proc. Natl. Acad. Sci., 1 April 2003.)