DOE Human Genome Program Contractor-Grantee
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
Microfabrication technology is an effective method for creating integrated devices for chemical and biochemical analysis1-3. 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/ÁL 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.
|The online presentation of this publication is a special feature of the Human Genome Project Information Web site.|