Analysis of Clinically Relevant Single-Nucleotide Polymorphisms by Use of Microelectronic Array Technology
Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the...
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Veröffentlicht in: | Clinical chemistry (Baltimore, Md.) Md.), 2002-12, Vol.48 (12), p.2124-2130 |
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Zusammenfassung: | Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes.
Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products.
Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods.
The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods. |
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ISSN: | 0009-9147 1530-8561 |
DOI: | 10.1093/clinchem/48.12.2124 |