Genotyping on a thermal gradient DNA chip.
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.
Kajiyama
Tomoharu
T
Department of Pediatrics, The Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Miyahara
Yuji
Y
Kricka
Larry J
LJ
Wilding
Peter
P
Graves
David J
DJ
Surrey
Saul
S
Fortina
Paolo
P
D005167:Factor VII / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005838:Genotype; D006361:Heating; D006801:Humans; D015336:Molecular Probe Techniques / Q000295:instrumentation; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017655:Silicon Compounds / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D013696:Temperature
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D017655:Silicon Compounds; D012822:Silicon Dioxide; D005167:Factor VII; C032734:silicon nitride