FUNCTIONAL-INTEGRATION OF PCR AMPLIFICATION AND CAPILLARY ELECTROPHORESIS IN A MICROFABRICATED DNA ANALYSIS DEVICE

Microfabricated silicon PCR reactors and glass capillary electrophores is (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling ca pabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (<120 s) DNA separations pro vided by microfabricated CE chips. The PCR chamber and the CE chip wer e directly linked through a photolithographically fabricated channel f illed with hydroxyethylcellulose sieving matrix. Electrophoretic injec tion directly from the PCR chamber through the cross injection channel was used as an ''electrophoretic valve'' to couple the PCR and CE dev ices oil-chip. To demonstrate the functionality of this system, a 15 m in PCR amplification of a beta-globin target cloned in M13 was immedia tely followed by high-speed CE chip separation in under 120 s, providi ng a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that chall enging amplifications of diagnostically interesting targets can also b e performed, Real-time monitoring df PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta -globin target as a function of cycle number was directly monitored fo r two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DMA analyses in microfabricated integrated fluidic systems.