Analytical validation of the Tag-It high-throughput microsphere-based universal array genotyping platform: Application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms

Abstract

Background: We have developed a novel, microsphere-based universal array platform referred to as the Tag-ItTM platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs). Methods: Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers. The products were then sorted on the Tag-It array and detected by use of the Luminex xMAPTM system. Genotypes were also determined by sequencing. Results: Empirical validation of the universal array showed that the highest nonspecific signal was 3.7% of the specific signal. Patient genotypes showed 100% concordance with direct DNA sequencing data for 736 SNP determinations. Conclusions: The Tag-It microsphere-based universal array platform is a highly accurate, multiplexed, high-throughput SNP-detection platform. © 2004 American Association for Clinical Chemistry.