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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. The simultaneous measurement in real time of the hybridization and melting on the entire oligonucleotide array was carried out with a fluorescence microscope equipped with CCD camera. The monitoring of the hybridization specificity for duplexes with different stabilities and AT content was enhanced by its measurement at optimal, discrimination temperatures on melting curves. Microchip diagnostics were optimized by choosing the proper allele-specific oligonucleotides from among the set of overlapping oligomers. The accuracy of mutation detection can be increased by simultaneous hybridization of the microchip with two differently labeled samples and by parallel monitoring their hybridization with a multi-wavelength fluorescence microscope. The efficiency and reliability of the sequence analysis were demonstrated with diagnostics for beta-thalassemia mutations.

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. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.