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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.

Oligonucleotide Microchip is a Versatile Tool for DNA Analysis: Diagnostic Applications

Igor Ivanov, Eugene Kirillov, Victor E.Barsky, Edward Kreindlin, Sergei Parinov, Edward Timofeev, Gennady Yershov, Vladimir L. Florentiev, and Andrei D. Mirzabekov
V.A Engelhardt Institute of Molecular Biology, 32 Vavilov Str., B-334, Moscow, 117984, Russia

Sequencing by Hybridization (SBH) is a kind of DNA technology based on the assumption to extract information about DNA sequence by specific hybridization with a set of oligonucleotides [1]. The architecture and complexity of the set defines the information capability. For example, hundreds oligonucleotides could be enough for diagnostics purposes and polymorphism analysis. These oligonucleotides can be fixed or even synthesized on the matrix and then hybridized with DNA.

We continue to develop the microchip DNA technology characterized by immobilization of oligonucleotides into small "cells" (upto 30x30 µm) of gel-support fixed on the glass plate [2]. After hybridization with DNA, fluorescent signals from each "cell" are detected and analyzed by specially designed microscope with CCD camera. Porous 3D structure of the polyacrylamide gel ensures beneficial properties for hybridization and washing out kinetics. The high capacity of immobilization oligonucleotides on gel-support and high hybridization capacity allow also using intercalating dyes for detecting perfect duplexes. In parallel with "standard" hybridization DNA to short oligonucleotides, "contiguous stacking" hybridization has been developed. In the latter case, fluorescently labeled pentamers are hybridized in juxtaposed position to the duplex formed between DNA and immobilized oligos. It has been shown that only perfect "contiguous stacked duplexes" are detected.

We have examined microchip technology in the model diagnostic experiments to identify mutations in blood samples of ß-thalassemia patients. The reliable discrimination of point mutations has been achieved by hybridization fluorescently tagged DNA with a set of octamers and decamers. We are showing the possibilities to apply this technology for diagnostics of any mutations along the whole gene length and for studying gene polymorphism.

This work was supported by grants from Russian Human Genome Project, U.S. Department of Energy and Affimax Research Institute.

[1.] A.D.Mirzabekov, DNA sequencing by hybridization - a megasequencing method and a diagnostic tool? Trends in Biotechnology (1994), vol.12, pp.27-32.
[2.] K.R.Khrapko et al., A method for DNA sequencing by hybridization with oligonucleotide matrix, J.Sequencing and Mapping (1991), vol.1, pp.375-388.