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Human Genome News Archive Edition

Human Genome News, March 1992; 3(6)

Moscow Workshop on Sequencing by Hybridization

A workshop on Sequencing by Hybridization (SBH) was held on November 19-20, 1991, at the Englehardt Institute of Molecular Biology in Moscow. Organized by the Human Genome Organization, the workshop was sponsored by DOE, the Wellcome Trust, and the Human Genome Project of the former U.S.S.R. The 44 participants, who came from the U.S.S.R., the United States, the United Kingdom, and Sweden, represented government and university research laboratories and several large and small companies.

The meeting was planned by Charles Cantor (Lawrence Berkeley Laboratory), Edwin Southern (Oxford University), and Andre Mirzabekov (Englehardt Institute of Molecular Biology).

SBH, developed independently by several research teams, is a set of related technologies that potentially could determine DNA sequence 100 or more times faster than now possible. Two formats are being developed for SBH:

  1. A single oligonucleotide probe is used to examine an array of sample DNAs immobilized on a surface.
  2. A single sample is hybridized to an immobilized matrix of oligonucleotides of overlapping sequence (a chip). If a chip for sequencing hundreds of thousands of nucleotides can be made inexpensively and if the sequencing procedure can be automated, the sequencing rate using SBH could approach millions of bases per day.

Meeting participants generally agreed that one or both SBH formats could develop into a useful sequencing tool in the near future, that appropriate combinations of conventional gel sequencing and SBH can be more efficient for genome sequencing than any one method, and that Format 1 can probably be implemented immediately.

The major obstacle to using SBH for sequencing is the lack of a full description of any sequence-dependent anomalies in short oligonucleotide interactions. Overcoming this obstacle will require parallel studies to generate large volumes of data. Such studies might simultaneously determine optimums for factors such as the surface to which samples are attached, the way the sample is immobilized, kinds of samples, hybridization conditions, and the manner in which hybridization is detected.

Potential ambiguities in SBH require that data-analysis algorithms produce statistical estimates of the likelihood that particular sequences are consistent with available data. Work with SBH technologies may actually put such data-analysis tools in place before they are required for more conventional DNA sequencing.

Due to the expense of synthesizing numerous oligomers, the initial cost of full-scale SBH implementation will be a high percentage of expected overall costs. Participants felt that international collaboration would greatly benefit the field and recommended long-term cooperation in sharing raw data and software, facilitating scientific exchange visits, and establishing an annual workshop to assess progress and advance technology. As work proceeds, sharing of oligonucleotide samples and arrays should help to reduce cost and effort and facilitate comparisons of the efficacy of various SBH implementations. Careful integration and continual assessment of SBH development during this expensive early stage are important.

All potential uses of SBH may not be fully envisioned yet, but its value in sequence comparisons and clinical diagnostics seems clear. Funding parallel efforts in the many aspects of SBH now, and coalescing successful developments into a more unified approach later, would avoid costly premature specialization.

Meeting Highlights

Hans Lehrach (Imperial Cancer Research Fund, London) presented a mapping strategy for linkup of cosmids through oligonucleotide fingerprint matching. He announced that ordering of the Schizosaccharomyces pombe genome in cosmids is imminent; the major role of oligonucleotide hybridization in this inquiry was emphasized.

In a pilot test with several related but unknown sequences, Radomir Crkvenjakov and Radoje Drmanac (Argonne National Laboratory) demonstrated that SBH can produce correct DNA sequence de novo. No wrong bases were called in 343 bp of hybridization-determined sequence.

Drmanac and Crkvenjakov (using Format 1) and Mirzabekov (using Format 2 in a gel) demonstrated the ability to distinguish between the hybridization of very short oligonucleotides to the complementary sequences and the hybridization of mismatched sequences or nonspecific binding to physical supports.

Drmanac presented results on the development of a hybridization data production line based on M13 clone libraries; 13,824 dots were made on an 8- by 12-cm filter by off-set printing of samples from 144 microtiter plates. Parallel clone growth and robotic spotting on filters in dense arrays will allow collection of up to 10 million clone-probe scores per day.

Southern described his laboratory's production of DNA probe arrays by an on-chip strategy that yields a complete array of 4s s-mers in s cycles (e.g., 65,536 octamers in 8 cycles). The Oxford group has made arrays of 4096 oligonucleotides on plates 20 by 20 cm.

Stephen Fodor described the approach being pursued by Affymax Research Corp., which uses addressable laser-activated photodeprotection in the chemical synthesis of oligonucleotides (or peptides) directly on a glass surface. Affymax scientists have recently developed new phosphoramidite derivatives capable of highly efficient light-activated detritylation and will now be evaluating these reagents for producing miniaturized DNA chips, including an octamer chip within a 1-square-in. area.

The Moscow group led by Mirzabekov is developing novel technologies for commercial production of sequencing chips that contain tens of immobilized oligonucleotides. They expect to produce chips with hundreds of oligonucleotides soon. Modified microelectronic technologies should make possible the production of thousands of chips containing hundreds of thousands of immobilized oligonucleotides costing $1.50 each.

Participants agreed on the need to explore the relative merits of hybridization detection by radioisotope decay, fluorescence, dielectric properties, and mass spectrometry.

For SBH Format 2, a major difficulty to be overcome is the need for more-complex chips, normalized matrices to compensate for the different stabilities of A-T and G-C base pairs in DNA duplexes, and methods for producing DNA fragments of narrow size distribution to avoid formation of secondary structures that interfere with hybridization.

Highlights of the informatics section of the conference were new ideas for modeling hybridization, fragment reconstruction, and sequencing chip design. Development of efficient software to unify mathematical and heuristic solutions and optimize parameters in comprehensive simulation experiments in the megabase range is visualized as the next step in SBH informatics.

John Elder (Oxford University) presented a linear model for going from sequence to detected SBH signal using data from Southern's chip. This general model and the associated parameter-estimation techniques provide a solid basis for further development of the hybridization models.

In summary, meeting participants felt that the Moscow SBH workshop was unusually stimulating, and investigators left the meeting firmly committed to this technology.


Workshop organizers collaborated to report on the meeting:

  • Charles Cantor
    Lawrence Berkeley Laboratory
    Principal Scientist of the DOE Human Genome Program
  • Edwin Southern
    Oxford University
  • Andre Mirzabekov
    Englehardt Institute of Molecular Biology

The full report of this meeting will be published in Genomics later this year.


HGMIS Staff

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