Multiplex genotyping of hereditary thrombophilia SNPs by SNaPshot technique

Abstract

Relevance: Testing of hereditary predisposition to thrombosis is an actual topic in medical genetic counseling. Information about increased risk for clotting abnormalities is important for preventing pregnancy complications, deep venous thrombosis, thromboembolism and other life-threatening conditions. Aim: To develop set of oligonucleotide probes for multiplex genotyping of several polymorphisms in the genes influencing thrombosis risk, using SNaPshot minisequencing technique. Materials and methods: The list of polymorphisms consisted of rs6025, rs6027, rs 1800595 in F5 ; rs1799963 in F2 ; rs5918 in ITGB3. For selecting the probes and PCR primers, Vector NTI и Primer3 programs were used. Results: After picking up the probes, possibility of multiplexing the polymorphisms in one tube was confirmed by Primer Focus kit reaction. Then several DNA samples with known genotypes were genotyped using the probes. In all cases, the genotypes determined by SNaPshot method corresponded to the genotypes detected by other methods. Conclusions: Panel of oligonucleotide probes for multiplex genotyping of five polymorphisms in the genes for coagulation factors II and V, and for integrin beta-3 (rs1799963, rs6025, rs6027, rs1800595, rs5918), has been developed and verified. The method allows speeding up and automatization of the genotyping process.