Functional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Device

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Abstract

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 °C/s heating, 2.5 °C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an “electrophoretic valve” to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a β-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR−CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR−CE devices is also feasible. Amplification of the β-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 × 107 and 4 × 105 copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.

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