Use of a competitive probe in assay design for genotyping of the UGT1A1*28 microsatellite polymorphism by the SMart-amplification process

A46

The complexity of molecular diagnostic assays is a significant barrier to employment at point-of-care, despite the growing need for such technologies. We have developed a sensitive, accurate, rapid, and simple DNA amplification scheme that shows potential for translational medicine from pharmacogenomics-based drug discovery thru to point-of-care diagnostics. Called the SMart Amplification Process version 2 (SMAP-2), the method employs a unique primer design and background suppression technology to minimize background amplification uses Thermus aquaticus MutS (Taq MutS) that can amplify only target sequences from blood sample without thermocycling.
 This time we report that use of SMAP-2 for polymorphism determination of the UGT1A1*28 allele required a further ancillary approach for complete background suppression. The UGT1A1*28 allele is a microsatellite copy number polymorphism. This is the first reported SMAP-2 assay designed for genotyping genetic variations of microsatellites. We found that by the addition of a primer to the amplification reaction, called a competitive probe (CP), assay specificity could be significantly enhanced. Including sample preparation time, we could rapidly detect the UGT1A1*28 polymorphism within 60 minutes using a CP-enhanced SMAP-2 assay. To test our method, we compared results from PCR-sequencing and the CP-enhanced SMAP-2 assay on 116 human blood samples for UGT1A1*28 polymorphism and demonstrated perfect concordance. These results illustrate the versatility of SMAP-2 for molecular diagnostics and provide a new approach for enhancing SMAP-2 assay specificity.