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Improvement of Low-Density Microelectronic Array Technology to Characterize 14 Mutations/Single-Nucleotide Polymorphisms from Several Human Genes on a Large Scale

¹ Unita’ Operativa Citogenetica e Genetica, Azienda Ospedaliera Careggi, Florence, Italy;² Dipartimento Area Critica Medico Chirurgica, University of Florence, Florence, Italy

^aaddress correspondence to this author at: Unita’ Operativa Citogenetica e Genetica, Azienda Ospedaliera Careggi, Viale Morgagni 85, 50134 Florence, Italy; fax 39-055-4279686, e-mail genomica@ao-careggi.toscana.it

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Large-scale human genetic studies require new technologies to genotype several samples with relative ease, high accuracy, and reasonable costs. Among the available approaches, a microelectronic array technology has been developed for DNA hybridization analysis of mutations/single-nucleotide polymorphisms (SNPs) (1)(2)(3)(4). The microelectronic array system (NanoChip® Molecular Biology Workstation; Nanogen) produces a defined electric field that allows charged molecules, such as nucleic acids, to be transported to any test site, or pad, on the electronic chip (NanoChip cartridge). Electronic-based molecule addressing can rapidly achieve a high concentration of amplicons on each pad of the cartridge. Control of temperature allows use of an optimal thermal stringency to characterize a SNP/mutation in all 100 pads of a cartridge simultaneously (5)(6). A thin hydrogel permeation layer overlies the pads; the presence of avidin or streptavidin in this layer allows the binding of biotinylated PCR products.

Although the technology is attractive, only a few protocols for its use have been published (7)(8)(9)(10)(11). We describe the development, optimization, and validation of a high-throughput method for SNPs and mutations analysis that allows performance of 1372 characterizations on each chip.

We studied samples from 150 individuals for 14 SNPs/mutations previously characterized by standard methods (restriction analysis, automatic sequencing, and allelic discrimination). Genomic DNA was isolated from peripheral blood by use of the FlexiGene DNA reagent set (QIAGEN GmbH). We analyzed 14 DNA mutations/SNPs for a total of 2100 characterizations (homozygous wild type, n = 1367; heterozygous, n = 522; homozygous mutant, n = 211). The 14 nucleotide substitutions were SNPs/mutations involving the glycoprotein Ia (GpIa), glycoprotein IIIa (GpIIIa), follicle-stimulating hormone receptor (FSHR), hereditary hemochromatosis . . . [Full Text of this Article]

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