Sequencing by hybridization with the generic 6-mer oligonucleotide microarray: an advanced scheme for data processing.

DNA sequencing by hybridization was carried out with a microarray of all 4(6) = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucleotides immobilized in 100 x 100 x 20-microm polyacrylamide gel pads of the generic microchip were hybridized with fluorescently labeled ssDNA, providing perfect and mismatched duplexes. Melting curves were measured in parallel for all microchip duplexes with a fluorescence microscope equipped with CCD camera.

Advanced method for oligonucleotide deprotection.

A new procedure for rapid deprotection of synthetic oligodeoxynucleotides has been developed. While all known deprotection methods require purification to remove the residual protective groups (e.g.

Polymorphism analysis and gene detection by minisequencing on an array of gel-immobilized primers.

Two procedures, multibase and multiprimer, have been developed for single nucleotide extension of primers immobilized within polyacrylamide gel pads on a microchip. In the multibase assay, a primer is next to a polymorphic nucleotide; the nucleotide is identified by the specificity with which the primer incorporates fluorescently labeled dideoxyribo-nucleoside triphosphates. In the multiprimer assay, several primers containing different 3'-terminal nucleotides overlapping the variable nucleotide in DNA are used.

Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips.

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes.

Sequence analysis by hybridization with oligonucleotide microchip: identification of beta-thalassemia mutations.

Diagnostics for genetic diseases were run and sequence analysis of DNA was carried out by hybridization of RNA transcripts with oligonucleotide array microchips. Polyacrylamide gel pads (100 x 100 x 20 microm) were fixed on a glass slide of the microchip and contained allele-specific immobilized oligonucleotides (10-mers). The RNA transcripts of PCR-amplified genomic DNA were fluorescently labeled by enzymatic or chemical methods and hybridized with the microchips.

Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides.

A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.

Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel.

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA.

DNA analysis and diagnostics on oligonucleotide microchips.

We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing "microchips." The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate.