Separating DNA sequencing fragments without a sieving matrix

The possibility of separating appropriately labeled DNA fragments using fre e-flow capillary electrophoresis was predicted a few years ago based on sim ple theoretical arguments. Free-flow separation of double-stranded DNA (dsD NA) fragments in the 100-1000 base range was later demonstrated using a str eptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (s sDNA). The first 100 bases of a DNA sequencing reaction were read without a ny sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not re quire coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper condi tions. ELFSE thus offers an attractive potential alternative to polymer sol utions for DNA sequencing in capillaries and microchips.