Primer design for SNP genotyping based on allele-specific amplification-Application to organ transplantation pharmacogenomics

Diagnostic methods based on single nucleotide polymorphism (SNP) biomarkers are essential for the real adoption of personalized medicine. Allele specific amplification in a homogeneous format or combined to microarray hybridization are powerful approaches for SNP genotyping. However, primers must be properly selected to minimize cross-reactivity, dimer formation and nonspecific hybridization. This study presents a design workflow diagram for the selection of required oligonucleotides for multiplex assays. Based on thermodynamic restrictions, the oligonucleotide sets are chosen for a specific amplification of wild- and mutant-type templates. Design constraints include the structural stability of primer-template duplexes, template-probe duplexes and self-annealing complexes or hairpins for each targeted gene. The performance of the design algorithm was evaluated for the simultaneous genotyping of three SNPs related to immunosuppressive drugs administered after solid organ transplantation. The assayed polymorphisms were rs1045642 (ABCB1 gene), rs1801133 (MTHFR gene) and rs776746 (CYP3A5 gene). Candidates were confirmed by discriminating homozygote and heterozygote populations using a fluorescence solution method and two colorimetric microarray methods on polycarbonate chips. The analysis of patient samples provided excellent genotyping results compared to those obtained by a reference method. The study demonstrates that the development of the allele-specific methods as pharmacogenetic tools can be simplified.