Multiplex Genotyping for Thrombophilia-Associated SNPs by Universal Bead Arrays

This chapter describes a method for the multiplex analysis of six biallelic single nucleotide polymorphisms (SNPs) associated with thrombophilia. The method may, however, be adapted for the simultaneous analysis of up to 100 markers (50 biallelic SNPs) in a single reaction. In the method described, the targets of interest are amplified by single-tube multiplex PCR using six primer sets followed by single-tube multiplex allele-specific primer extension using 12 universally tagged genotyping primers. Labeled extension products are sorted using the xTAG™ universal bead-based array and detected on the Luminex xMAPˆledR system. The 12 universal tag sequences used in the assay derive from a set of 100 universal tags which have been designed to be isothermal and have been empirically validated to show that mismatch hybridization events are minimal. The method is suitable for cost-effective high-throughput clinical genotyping applications.