Toward real-world sequencing by microdevice electrophoresis.

Significant advancement in the technology of DNA analysis is expected from the use of microfabricated electrophoretic devices for sequencing and genotyping. In this approach photolithography, combined with wet-etching and thermal wafer bonding, is used to construct enclosed intricate microchannel structures in glass and fused-silica substrates; these structures are then utilized for electrophoresis (Harrison et al. 1993). It has been speculated that these devices will allow DNA separations approaching the theoretical limits of electrophoresis and in a format that will reduce analysis time and extend parallelism and automation (Freemantle 1999), which might hence increase throughput well beyond current capillary array machines. For example, in recent experiments we have demonstrated genotyping at 10- to 100-fold reduced analysis times on microdevices when compared to capillaries and slab gels, respectively (Schmalzing et al. 1997, 1999). DNA sequencing of single-color pGEM and four-color M13 DNA standard sequencing samples has been demonstrated on 3.5-, 11.5-, and 7-cm-long microdevices (Woolley et al. 1995; Schmalzing et al. 1998; Liu et al. 1999). The feasibility of ultra-high sample throughput has been proven through still modest multiplexing up to 96 microchannels (Simpson et al. 1998; Koutny et al. 1999). However, to the best of our knowledge, all published studies on DNA sequencing by microdevices have been performed using DNA standard samples such as M13 or pGEM. Practical sequencing must deal with additional factors such as variable salt and template concentrations (Ruiz-Martinez et al. 1998; Salas-Solano et al. 1998), highly sample-specific compression regions, and the interplay between electrophoretic separation and base-calling software typical of production DNA sequencing samples. We report initial results on how microdevices perform under practical conditions using DNA sequencing samples as prepared for high throughput, cost-sensitive sequencing under the Human Genome Project. Our results suggest that much of the anticipated throughput improvement for microdevice sequencing is feasible.