Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Electronically controlled DNA hybridization on semiconductor microchips: A miniaturized DNA chip format for rapid medical diagnostics and DNA sequencing.

Glen A. Evans[1,2], Harold R. Garner[1], Eugene Tu[2], William F. Butler[2], Ronald G. Sosnowski[2], Donald D. Montgomery[2] and Michael J. Heller[2].
[1]McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, Texas 75235; and [2]Nanogen, Incorporated, 11588 Sorrento Valley Road, San Diego, California 92121.

DNA hybridization is an important basic technique for molecular biology and medical diagnostics. We have developed a novel technology which allows multiplex DNA hybridization reactions to be carried out in microarray formats or the surface of a unique semiconductor device, or DNA microchip. This technology, termed APEX (for Active Programmable Electronic Matrix) utilizes microdevices containing arrays of electronically addressable microlocations on a silicon surface fabricated using microlithography. Arrays are designed such that direct electronic currents can be independently applied and controlled at each element on the array. Specific oligonucleotide probe sequences are covalently attached to the surface of metal microeletrodes, or to polymer chip coatings, and self-assembly can be directed by the microchip. A prototype APEX device (Nanogen 8850 Chip) has been manufactured and contains 64 addressable microlocations (50 µm x 50 µm) in a space of less than 1 mm[2] on a silicon/silicon dioxide substrate and metal electrodes that can be operated in a direct current mode. The device operates by placing a DNA sample over the array and applies positive bias to the underlying microelectrodes. Target DNA becomes immediately concentrated at each microlocation (by a factor of > 10(6)) through free field electrophoresis and, as a result of the electrophoretic concentration, the hybridization rate for the target probe and test probe is greatly increased. Reversing the polarity of the electric field allows the removal of non-hybridized DNA, while quantitative control of the reverse bias allows independent adjustment of hybridization stringency at each test site. Exquisite control of hybridization stringency conditions can be achieved at room temperature through electronic control. The hybridization signal of fluorescently-labeled target DNA is detected using CCD arrays or external cooled CCD cameras. This technique has a number of powerful and novel features: 1) it allows hybridization reactions to be programmed, carried out automatically, and results obtained within seconds; 2) it allows precise and independent control of hybridization stringency over each individual sequence in a hybridization array; 3) electronic control over each hybridization site allows highly selective single base miss-match discrimination to be achieved with probes ranging from 7 to 22 nucleotides. We have utilized APEX devices for clinical HLA typing as well as for other diagnostic and second generation chips with a larger number of elements are being developed for massive parallel DNA analysis for human genotyping and for sequencing by hybridization applications. This novel technique may have a variety of other research and commercial applications as well as special applications in the Human Genome Project.