Multiple Sample PCR Analysis

Previously, we have integrated the steps of thermal cell lysis to release DNA, simultaneous amplification of multiple gene loci using selected primer sets, and electrophoretic analysis of the amplified products on a single microchip. In the course of that work it was determined that many electrophoretic runs can be made in the same separation channel with a single filling of sieving medium. Furthermore, the analysis time is very short compared to the time that is currently required for sample DNA amplification. An obvious way to take advantage of these features and to increase throughput of genetic analysis is to increase the number of DNA samples to be amplified on a single microchip. Figure 1 illustrates the designs of a first generation multiple sample PCR microchip for the amplification and electrophoretic analysis of four DNA samples and of a sizing microchip for accurately assessing the sizes of the products made. The PCR products made in the 4 reaction wells can be analyzed individually or in combinations via the appropriate injection scheme. Figure 2 (a, b and c) shows the results obtained when three DNA samples, bacteriophage (500 bp), plasmid (410 bp) and bacteria (346 bp), are amplified in separate reaction wells and individually analyzed using the microchip design shown in Figure 1a. Figure 2(d) illustrates the simultaneous analysis of all three products by co-injecting from all three wells. In a similar manner four DNAs, including a 199-bp bacteriophage target, are amplified and analyzed (Figure 3). The sizes of the products are verified by co-electrophoresis with marker DNA (Figure 4) using the microchip illustrated in Figure 1b.

Future effort will be directed toward integrating different DNA sample preparation and analysis steps in a single microchip device. The goal is to adapt this microchip technology to the analysis of mammalian DNAs for research (functional genomics), forensics and medical diagnostics.

(1) L.C. Waters, S.C. Jacobson, N. Kroutchinina, J. Khandurina, R.S. Foote, and J.M. Ramsey, "Multiple Sample PCR Amplification and Electrophoretic Analysis on a Microchip," Analytical Chemistry, 70, 5172-5176, 1998

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