Multiplex PCR and CE Analysis Integrated on a Microchip

The ability to perform all of the steps of a biological assay on a single microchip promises significant advantages to biochemists and molecular biologists in terms of speed, cost and automation. The demonstration of a device that can perform multiplex polymerase chain reactions (PCR) and electrophoretic sizing is particularly interesting because of its potentially widespread application in molecular biology, clinical and forensics laboratories for high-throughput genetic analysis. The individual steps for performing complete multiplex PCR analysis of whole cells are readily adaptable to the microchip format. Toward this goal, 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.

Figure 1 shows the microchip designs used: (a) to amplify DNA and electrophoretically analyze the PCR product(s) on the same microchip and (b) to accurately size the PCR products made. Figure 2(a) is an electropherogram of the PCR products obtained by amplification of a 500 bp target of l DNA with amplification and capillary electrophoretic (CE) analysis done on the same microchip, i.e., design (a) shown in Figure 1. Figure 2(b) illustrates the sizing of those PCR products on a separate microchip, i.e., design (b) shown in Figure 1, by co-electrophoresis with a DNA sizing ladder, FX174 -Hae III digest.

Cell lysis, multiplexed PCR and electrophoretic analysis in a single microchip is demonstrated in Figure 3. Whole E. coli cells are thermally lysed in the PCR mixture during the first 6 min of the initial temperature setting of 94 C (design a, Figure 1). The subsequent PCR amplification is carried out using 9 primers (5 sets with 2 of the sets sharing a single primer). Following amplification, the products are electrokinetically transported to load the injection valve. The separation is performed using polydimethylacrylamide as the sieving medium. The first three peaks (154, 264, and 346 bp) are from the E. coli genomic DNA, and the last two peaks (410 and 550 bp) are from the plasmid target, plant gene agNt84.

Future effort will be directed toward expanding the number of different DNA samples that can be analyzed in this format. The goal is to adapt this microchip technology to the analysis of mammalian DNAs for research (functional genomics), forensics, and medical diagnostics.

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