DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

24. Integrated Microfluidic DNA Amplification and Analysis Systems

Eric T. Lagally¹, Daojing Wang², Charles Emrich², and Richard A. Mathies²

¹UCB/UCSF Joint Graduate Group in Bioengineering and ²Department of Chemistry, University of California, Berkeley, CA 94720

lagally@zinc.cchem.berkeley.edu

Microfabrication technology is an effective method for creating integrated devices for chemical and biochemical analysis^1-3. Our early work in the development of integrated devices included the manufacture of a hybrid Si polymerase chain reaction (PCR) reactor mated with a glass capillary electrophoresis (CE) device⁴ and the development of a CE device with an integrated electrochemical detector⁵. In more recent work, we have developed a fully integrated DNA analysis system microfabricated in glass consisting of controlled fluid delivery using active and passive elements, PCR amplification, and direct coupling to a capillary electrophoretic separation⁶. Samples are introduced at a common sample bus and loaded precisely into a 280-nanoliter volume PCR reactor using valves and hydrophobic vents. The sample is cycled between three temperatures using a resistive heater mounted on the bottom of the chip, and the amplification products are then directly injected and separated on a capillary electrophoresis channel. The device takes only 33 seconds/cycle, representing a vast improvement over conventional thermal cycling systems, which can take up to 5 minutes/cycle. Amplicons from the M13/pUC19 plasmid have been produced from only 20 starting copies/µL or 5 copies in the reactor. This amplification is among the most sensitive compared both to previous static systems, which require ~6,000 starting copies⁷, and to continuous-flow geometries which require as many as ~108 starting copies⁸. The high sensitivity of this device allows studies at the single molecule level.

We have also developed a microfluidic DNA capture chamber for sequencing sample clean-up and concentration. This device uses microfluidic elements to flow raw sequencing samples through a filter chamber filled with oligonucleotide-labeled capture beads. The extension products of interest are selectively captured on the beads and subsequently released using formamide and heat. The capture chamber is directly connected to a capillary electrophoresis channel for immediate sequencing. These results demonstrate a key link in the development of an integrated microfluidic system that performs complete genetic analyses at sub-microliter volumes.

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