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Genotyping on a Thermal Gradient DNA Chip

2003; Cold Spring Harbor Laboratory Press; Volume: 13; Issue: 3 Linguagem: Inglês

10.1101/gr.790603

1549-5469

Tomoharu Kajiyama, Yuji Miyahara, Larry J. Kricka, Peter Wilding, David J. Graves, Saul Surrey, Paolo Fortina,

Molecular Biology Techniques and Applications

Silicon-based chips with discrete, independently temperature-controlled islands have been developed for use in DNA microarray hybridization studies. Each island, containing a heater made of a diffusion layer and a temperature sensor based on a p–n junction, is created on a silicon dioxide/nitride surface by anisotropic etching. Different reactive groups are subsequently added to the surface of the islands, and allele-specific oligonucleotide probes are attached to discrete spots on the chip. Hybridization is performed with Cy5-tagged single-stranded targets derived by PCR from genomic DNA. Results are assessed by measuring fluorescence of bound dye-tagged targets after hybridization and washing. Temperatures at each island can be set at different values to obtain optimal distinction between perfect matches and mismatches. This approach facilitates definition of optimal temperatures for probe/target annealing and for distinction between perfectly matched versus mismatched solution-phase targets. The thermal gradient DNA chips were then tested for genotyping, and the results for four different loci in two genes are presented. Unambiguous typing was achieved for clinically relevant loci within the factor VII and hemochromatosis genes.