41. Integrated Microchip Devices for DNA Analysis 

R. S. Foote, W. C. Dunn, J. Khandurina, N. Kroutchinina, T. McKnight, L. C. Waters, S. C. Jacobson, and J. M. Ramsey 
Chemical & Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37830-6142 
footers@ornl.gov 

Microfabricated microfluidic devices are being developed for integrated processing and analysis of DNA samples. The steps of DNA extraction, amplification, preconcentration and electrophoretic analysis can be carried out on monolithic devices. We have previously demonstrated integrated cell lysis, multiplex PCR and capillary electrophoretic (CE) size analysis on microchips using bacterial samples. Integrated PCR-CE microchips are now being used for the analysis of simple sequence repeat (SSR) loci in mammalian genomes. To demonstrate the potential utility of this technology for rapid PCR-based gene mapping, SSR polymorphisms (SSRPs) at two mouse genome loci, D4Mit141 and D8Mit9, were identified and compared for Mus musculus (C57/Bl or C3H), Mus spretus, a C57/Bl x spretus hybrid, and progeny from an interspecies (C3H x spretus) backcross used to create genetic maps. For both loci, microchip electrophoresis patterns of SSR fragments generated from animals heterozygous for musculus and spretus alleles were clearly distinguishable from those of homozygotes and PCR product sizes were determined for respective SSRs by co-electrophoresis with marker DNA. Integrated microchip analysis of human forensic samples has also been demonstrated by DNA fingerprinting at the CSF1PO, THO1, TPOX and vWA loci. 

The microchip CE analysis time for PCR products is typically less than 5 minutes, so that the throughput for integrated PCR-CE microdevices is primarily determined by the PCR thermal cycling time. Approaches to increasing the throughput of these devices include the use of multiple reaction chambers for parallel PCR, fast thermal cycling using thermoelectric heating and cooling, and on-chip concentration of products from low cycle number PCRs. The last approach is being explored by incorporating a DNA concentration region into the microchip architecture between the reaction chamber and the separation channel. A porous membrane between two parallel channels is incorporated into the channel manifold using a silicate adhesive to bond the cover plate to the substrate. The thin silicate layer serves as a semi-permeable membrane allowing ionic current to pass between the separated channels but retaining large DNA molecules. Preconcentrated sample is then injected into the separation channel and electrophoretically analyzed. DNA fragments were concentrated on-chip from PCR amplifications by up to 2 orders of magnitude by this method, allowing product analysis at a reduced number of thermal cycles.