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Human Genome News Archive Edition

Human Genome News, January 1994; 5(5)

Workshop on Sequencing by Hybridization

The Second International Workshop on Sequencing by Hybridization (SBH)was held at the Houston Advanced Research Center (HARC) in The Woodlands, Texas, on October 28-30, 1993. The workshop was organized by Kenneth Beattie (HARC) and supported by DOE; the Human Genome Organization; The Wellcome Trust; HARC; Beckman Instruments, Inc.; Affymetrix, Inc.; and Genosys Biotechnologies, Inc. Some 75 scientists from 8 countries attended the meeting, which reported significant progress in the SBH field since the inaugural workshop held 2 years ago in Moscow. Some highlights are summarized below.

Chemistry and Analogs
In a session chaired by Daniel Brown (Medical Research Council, Cambridge, U.K.), recent advances were reviewed in chemical synthesis, modification, and surface attachment of hybridization probes. Gerald Hurst (Genosys Biotechnologies) opened the conference with a discussion of high-throughput oligonucleotide synthesis. Robert Matson and Peter Coassin (Beckman Instruments)reported the hybridization properties of oligonucleotide arrays synthesized directly on polypropylene sheets. Philip Andrews (University of Michigan) discussed the chemical synthesis and hybridization properties of improved universal base analogs, which potentially can be incorporated into gapped probes to extend the length of DNA sequence that can be reconstructed from SBH data. Stephen Case-Green (Oxford University) presented data on the positional effects of mispairing. The analog inosine (and probably canonical nucleosides), for example, tends to base pair with reduced specificity during hybridization when present at the ends of a duplex, especially the 3' end. Andrei Mirzabekov (Engelhardt Institute of Molecular Biology, Moscow) reviewed the significant progress of his laboratory in constructing hybridization matrices within thin films of polyacrylamide. Roger Giese (Northeastern University) ended the session with a description of electrophore DNA labels, which could be coupled with electron-capture mass spectrometry to enable a vast multiplexing of hybridization reactions.

Engineering and Automation
Progress in the production of hybridization arrays was summarized in the session chaired by Mirzabekov. Stephen Fodor (Affymetrix, Inc.) reviewed the impressive progress made in the use of photolithography to synthesize gene-targeted oligonucleotide arrays directly onto silicon dioxide substrates and described the use of these arrays to analyze mutations in cystic fibrosis and p53 exons. Several speakers described microfabricated genosensors for use in SBH. Dennis Rathman [Massachusetts Institute of Technology (MIT)] reported recent progress in developing an electronic permittivity device for direct detection of hybridization within a genosensor array; Robert Reich (MIT) described a charge-coupled device genosensor for direct-contact imaging of hybridization; and Beattie introduced the concept of a flow-through genosensor designed for improved detection sensitivity and the ability to analyze dilute nucleic acid solutions. David Wallace (Microfab Technologies, Inc.) described a microfluidic jet system being developed for precision dispensing of DNA solutions to individual test sites within hybridization arrays.

Victor Barsky and Gennady Yershov (Engelhardt Institute) described engineering aspects of preparing gel matrix sequencing microchips. Edwin Southern (Oxford University) presented a discussion of RNA analysis by hybridization to a novel oligonucleotide array synthesized directly onto glass. Hybridization data were not predicted entirely from the higher-order structure of target RNA nor from using energy calculations. Southern suggested that scanning arrays could be useful for optimizing primer selection for polymerase chain reactions as well as for antisense oligonucleotide design and mutation detection.

Hybridization Data
Uwe Maskos (NIH) opened a session chaired by Southern with a forthright discussion of the challenges of interpreting real data obtained by hybridization of target DNA with oligonucleotide arrays. Using representative data obtained in his extensive work in Southern's laboratory, Maskos stressed that the SBH community must learn to deal with hybridization patterns that are only partially predicted from a known target sequence. A vast hybridization data set should be acquired, he continued, to enable definition of hybridization rules that will be needed for valid data interpretation and for guiding the selection of appropriate probes to include in an array. Toward this end, Mitch Doktycz [Oak Ridge National Laboratory (ORNL)] gave a progress report on an extensive effort to catalog the effects of base sequence on octamer hybridization in solution. This work, to be repeated later in surface hybridization in collaboration with Robert Foote (ORNL) and Beattie, will examine mismatch discrimination as a function of sequence and position.

James Wetmur (Mount Sinai School of Medicine) emphasized the importance of the Doktycz approach and presented a theory for thermodynamic and kinetic consequences of stacking and branching in SBH. Data on tandem ligation strategies were presented by Levy Ulanovsky (Weizmann Institute, Israel), Keith Kretz (Stratagene), and Cassandra Smith (Boston University). Zhen Guo (University of Wisconsin) reported optimization of linker arm and attachment density in surface hybridization and presented results on detecting mutations in the human tyrosinase gene.

Elmar Maier and Sabastian Meier-Ewert (Imperial Cancer Research Fund, London), Jorg Hoheisel (German Cancer Research Center), and Radomir Crkvenjakov and Radoje Drmanac [Argonne National Laboratory (ANL)] summarized the use of robotics to prepare very large numbers of DNA samples and array them at high density onto membranes. Real progress was also reported by these investigators in the use of such arrays in clone mapping and partial sequencing.

Theory and Informatics
Crkvenjakov chaired a session that focused on the usefulness of reduced probe sets and additional biochemical data in SBH. William Bains (PA Consulting Group and Cambridge Laboratory) discussed the utility of a reduced-set hybridization chip containing 4096 octamers with 2 redundant sites and also pointed out that analysis of cDNAs by SBH is facilitated when a single open reading frame is known. Alexander Chetverin (Institute of Protein Research, Moscow Region) described an elaborate nested-strand hybridization strategy that may enable sequencing of complex mixtures of fragments. SBH strategies for recognizing functional gene regions in genomic DNA were discussed by Victor Solovyev (Baylor College of Medicine) and Ivan Labat (ANL).

Yuri Lysov (Engelhardt Institute) described a computer simulation with additional rounds of continuous stacking hybridization to extend the length of sequences that can be determined by SBH. Pavel Pevzner (Pennsylvania State University) described binary chips requiring 5 to 10% of the probe number. Aleksandar Milosavljevic (ANL) and Robert Lipshutz (Affymetrix) described comprehensive sets of software tools that have been developed to handle the informatics needs of large SBH projects.

Kenneth Beattie, HARC

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