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Human Genome News Archive Edition

Human Genome News, January 1993; 4(5)

DNA Chip Promises Faster Sequencing

International Cooperation Among Industry, Academia, National Laboratories Leads to New Sequencing Technology Development

The NIH National Center for Human Genome Research has awarded 3-year, $2-million grants to the Houston Advanced Research Center (HARC) in Houston, Texas, and the Affymax Research Institute in Palo Alto, California, to develop sequencing by hybridization (SBH) technologies. These technologies, which have the potential to increase DNA sequencing rates 100 or more times, are based on the identification of target sequences by their complementary binding to oligonucleotide probes on an immobilized matrix, called a chip.

HARC, an independent, nonprofit research organization fostering scientific and technological research and emphasizing technology transfer, will oversee the design, fabrication, and testing of chips ("genosensors"). Affymax will apply VLSIPS (very large scale immobilized polymer synthesis) chip technology to DNA sequencing. Kenneth Beattie heads the multidisciplinary HARC team that also includes Mitchell Eggers, chemists from the Baylor College of Medicine Center for Biotechnology led by Michael Hogan, and a microfabrication team led by Daniel Ehrlich at the Massachusetts Institute of Technology. The Affymax group, led by Stephen Fodor, includes Robert Lipshutz (Wagner Associates), Ronald Davis (Stanford University School of Medicine), and Pavel Pevzner (Pennsylvania State University).

SBH Principles and Challenges

The SBH concept was independently conceived 4 to 5 years ago by several European scientists- Radomir Crkvenjakov and Radoje Drmanac in Belgrade, Yugoslavia (now at Argonne National Laboratory), Edwin Southern and William Baines in the United Kingdom, and the group headed by Andrei Mirzabekov at Englehardt Institute in Moscow. In principle, hybridization of a target sequence with a complete set of all probes of a given length [e.g., all 65,536 (4 to the 8th) octamers] can reveal the complete oligonucleotide content of the DNA sample; this information is input to computational algorithms that output extended DNA sequence. Each chip is expected to contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.

Both DOE and NIH have invested in this emerging method, which is gaining widespread recognition for its potential use in genetic analysis, especially in sequence comparisons (diagnostics and polymorphic marker analysis). SBH technology involves many technical challenges, among them multiple occurrences of probe sequences within the target, promotion of highly accurate hybridization over a wide range of base compositions, preparation of miniaturized DNA arrays, sensitive detection of hybridization, computer processing of hybridization data, and automated manipulation of numerous DNA samples.

HARC Strategies

The developmental grant to HARC supports work in four project components:

  • attachment of oligonucleotide probes to surfaces via a variety of linker arms;
  • detection of hybridization by permittivity measurements within microfabricated sample wells containing microelectrodes and surface-bound probes (capture of long target strands by the short surface-bound probes elicits a change in the electrical properties within a sample well);
  • development of initial SBH applications for genome mapping; and
  • establishment of a program to make SBH broadly available within the research community through a variety of methods, including an electronic information network.

The team will develop means for microrobotic placement of oligonucleotides to form a miniaturized chip and develop computational tools for analyzing hybridization data. HARC has initiated collaborations with research groups led by Robert Foote and Bruce Jacobson (Oak Ridge National Laboratory) and Mirzabekov. HARC's collaboration with Mirzabekov's group is being supported by a grant from the International Genome Research Collaborative Program sponsored by NCHGR.

Affymax Strategies

Long-term research goals at Affymax are (1) construction of spatially defined arrays of oligonucleotide probes by applying newly developed techniques in light-directed polymer synthesis to oligonucleotides and (2) assessment of the feasibility of using these arrays in SBH. This effort requires the three developments described below.

An efficient, low-cost strategy for generating large arrays of oligonucleotides using photolithographic techniques and solid-phase chemical synthesis. Initially developed at Affymax to synthesize peptides on a glass surface for protein recognition, this approach substitutes a photoremovable protecting group for the standard acid-labile dimethoxytrityl groups at the 5'-hydroxyl coupling site in standard phosphoramidite synthesis. This allows integration of photolithography, miniaturization, and combinatorial synthesis methods into the solid-phase synthesis cycle.

A detection technology capable of assaying hybridization of target molecules to surface-bound oligonucleotides. Affymax researchers hope to achieve this goal by tagging target DNA with fluorescent reporter groups and then reading the array with epifluorescence microscopy. This approach will allow assessment of hybridization kinetics, identification of the hybrid on the array, and real-time analysis of target DNA-oligomer disassociation behavior.

Algorithms to interpret the results of hybridization experiments. Affymax will attempt to develop efficient applications for sequencing, sequence checking, physical mapping, functional mapping, homology search, and the detection of introns and exons.

Affymax is also funded as a Small Business Innovation Research project in the DOE Human Genome Program.

Human Genome News 3(6), 12-13, 16 (March 1992) included a report on the international SBH workshop held in Moscow in November 1991. The next SBH international workshop is tentatively scheduled for October of this year. [Contact: Kenneth Beattie; HARC; The Woodlands, Tx 77381 (713/363-7947, Fax: -7914, Internet:]

The DNA sequencing "chip" is a solid substrate comprising an array of many short DNA pieces (in this example octamers) having known, overlapping sequences. A longer sample DNA of unknown sequence will bind (A to T, C to G) to chip octamers that have sequences complementary to particular 8-base segments on the sample. The specific binding pattern can be read by a computer, and the resulting nested octamer set is used to determine the sequence of the sample.

Reported by Denise Casey
HGMIS, Oak Ridge National Laboratory

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Human Genome Project 1990–2003

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