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

Abstract

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.

Estimation of Warfarin Maintenance Dose Based on VKORC1 (−1639 G>A) and CYP2C9 Genotypes

“…Thus 1 critical operational aspect is for there to be clear differentiation between the allelic ratios obtained for each of the potential genotype determinations. As is true for other assays that we have developed with this technology (16 ), this assay demonstrates a high degree of precision in the allelic ratio for each nucleotide position tested by the method, thus yielding a high degree of certainty with regard to differentiation of the genotype. Furthermore, through the analysis of a variety of synthetic and genomic DNA samples, providing a diverse array of possible genotype structures, we have demonstrated the high degree of accuracy that can be achieved with this technology.…”

“…We simultaneously detected CYP2C9*2 (rs1799853), *3 (rs1057910), *4, *5 (rs28371686), and *6 (rs9332131) alleles and VKORC1 Ϫ1639GϾA (rs9923231), ϩ85GϾT, ϩ121GϾT, ϩ134TϾC, ϩ172AϾG, ϩ1331GϾA, and ϩ3487TϾG variants by use of the 2C9ϩVKORC1 Mutation Detection Kit as indicated in the manufacturer's product label (formerly Tm Bioscience, now Luminex Molecular Diagnostics) (15,16 ). Detection of the CYP2C9*1 allele is based on the absence of all CYP2C9 nucleotide changes detected by the assay.…”

“…After hybridization and washing, we incubated extended allele-specific primers containing biotinylated cytosine in the presence of streptavidin-R-phycoerythrin (Molecular Probes). Finally, we sorted beads on the basis of fluorescence and measured the median fluorescence intensity (MFI) of R-phycoerythrin by use of a Luminex ® 100 TM System (16 ).…”

Application of a Microsphere-Based Array for Rapid Identification of Acinetobacter spp. with Distinct Antimicrobial Susceptibilities

“…The ASPE assay is based on the design of a single-nucleotide polymorphism (SNP)-specific nucleotide at the 3Ј end of each extension primer and can readily discriminate any SNP or mutant. The utilization of the ZipCode/cZipCode hybridization enabled universal hybridization with microspheres (29,36). The cZipCode-coupled beads also can be used in other DNA-based tests, which will economize bead usage.…”

“…The microsphere-based array provides the capacity for conducting up to 100 biological reactions simultaneously in a single reaction vessel, and it combines the specificity and reliability of oligonucleotide hybridization analysis with the speed and sensitivity of a flow cytometer (36). Furthermore, this method has been applied reliably to species identification (23), the genotyping (6) of pathogens, and mutation detection (29).…”