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This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: clinchem.2005.063198v1 52/6/1033    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Zhu, Y. Articles by Linder, M. W. Search for Related Content PubMed PubMed Citation Articles by Zhu, Y. Articles by Linder, M. W. Related Collections Molecular Diagnostics and Genetics Drug Monitoring and Toxicology Automation and Analytical Techniques

Simultaneous Determination of 7 N-Acetyltransferase-2 Single-Nucleotide Variations by Allele-Specific Primer Extension Assay

Departments of¹ Pathology and Laboratory Medicine and ² Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY.

^aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY 40202. Fax 502-852-1177; e-mail mwlind01{at}gwise.louisville.edu.

Background: Genotyping of N-acetyltransferase-2 (NAT2) is useful in predicting the risk for toxicity of NAT2 substrates. Current methods cannot detect the 7 most important single-nucleotide variations in NAT2 simultaneously in 1 tube.

Methods: We developed an assay that uses allele-specific primer extension (ASPE) and microsphere hybridization for the simultaneous detection of 7 single-nucleotide variations in NAT2. Using 12 samples previously genotyped by a TaqMan-based assay for method development and as positive controls, we amplified the genetic locus of NAT2 comprising the single-nucleotide variations of interest by PCR and then performed ASPE with allele-specific primers and biotinylated dCTP followed by bead hybridization and streptavidin–R-phycoerythrin binding. Genotypes were determined according to the allele-specific fluorescent signal ratios.

Results: The mean (SD) allelic ratios for homozygous common, heterozygous variant, and homozygous variant NAT2 genotypes were 0.0394 (0.0113) (n = 80), 0.4372 (0.0270) (n = 148), and 0.9331 (0.0127) (n = 325). The assay had 100% (95% confidence interval, 99%–100%) within-run reproducibility for 12 samples repeated 6 times and 100% (98%–100%) between-run reproducibility for a 5-sample subset run on 6 different days. NAT2 genotypes of 30 blinded samples determined by this assay were 100% (98%–100%) concordant with results obtained using the TaqMan method.

Conclusions: The developed assay can simultaneously determine single-nucleotide variations in NAT2. The assay demonstrates no overlap in allele-specific signal ratios between homozygous common, heterozygous, and homozygous variant and shows agreement with a reference method and reproducibility of genotype identification.

The following articles in journals at HighWire Press have cited this article:

				J. A. G. Agundez, K. Golka, C. Martinez, S. Selinski, M. Blaszkewicz, and E. Garcia-Martin Unraveling Ambiguous NAT2 Genotyping Data Clin. Chem., August 1, 2008; 54(8): 1390 - 1394. [Abstract] [Full Text] [PDF]

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