Detection of blood group genes using multiplex SNaPshot method

The determination of blood group antigens in patients and donors is of primary importance in transfusion medicine. Blood group antigens are inherited and are polymorphic in nature. The majority of polymorphic blood group antigens arise from single-nucleotide polymorphisms (SNPs) in the blood group genes. Many DNA-based assays, such as species-specific polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and microchips, have been described to study variant blood group genes. In this study, the SNaPshot (Applied Biosystems) method was adapted to detect SNPs in 10 common blood group systems.

DNA regions of interest were amplified in multiplex PCR and annealed to specific oligonucleotide probe primers of different lengths. AmpliTaq DNA polymerase extended the primers by adding only a single fluorescent ddNTP to its 3' end and was detected by differential mobility in capillary electrophoresis in a genetic analyzer. Results were analyzed using computer software in SNaPshot default analysis method.

Seventeen SNP sites in 29 blood samples, previously phenotyped and/or genotyped, were used to test the accuracy and reproducibility of multiplex SNaPshot assays. The results were compared with the previously analyzed types. SNaPshot analyses predicted the 17 SNP sites accurately for all the 29 blood samples. Both homozygous and heterozygous blood groups were detected with equal confidence.

Blood group detection by SNaPshot method is a practical alternative to antibody-dependent phenotype prediction. Starting with DNA, this method is fast with a turnaround time of 24 hours with mean reagent cost around $2 per SNP detected.