Rapid Detection of 871G>A Mutation by Sequence Specific PCR in ABO*A301 Blood Donors.

Background: A3 blood subgroup is heterogeneous at molecular level. To date only two cases of ABO*A301 allele containing a single substitution 871G>A (Asp291Asn) were found in A3B individuals exclusively. The purpose of this study was to use a sequence specific PCR (PCR-SSP) to detect 871G>A mutation in A3 and A3B samples.

Material and methods: Twenty blood samples from unrelated donors were classified serologically as A3 and A3B using tube test technique and gel-test simultaneously. Agglutination of red cells by lectins was performed according to manufacturer’s instructions using anti-A1 lectin from Dolichos biflorus and anti-H from Ulex europaeus. Genomic DNA was prepared from peripheral blood cells by a salting-out method. ABO genotyping was performed using a polymerase chain reaction method with sequence specific primers: 95s (5′-CAC CAG GCC ATG ATG GTC A-3′) for consense G detection, 96s (5′-CAC CAG GCC ATG ATG GTC A-3′) for mutation A detection and 244as (5′-CGC AGT GAA CCT CAG CTT C-3′) for both detections on gene ABO exon VII (Seltsam, Transfusion 2003, 43(4): 428-39). PCR amplification was carried out under the following conditions: 5 initial cycles at 94°C for 10 minutes and 65°C for 1 minute; 20 cycles of 94°C for 10 seconds and 65°C for 1 minute; 20 final cycles of 94°C for 20 seconds, 61.5°C for 50 seconds and 72°C for 30 seconds. PCR amplification of 0.1 μg of genomic DNA was performed in a total volume of 25 μL containing 0.2 mM of each dNTP, 5 units Taq DNA polymerase in the buffer supplied, 2.5 mM MgCl₂ and 12.5 pmoles of each primer. Fragment of 177 bp derived from each mutation or consense assay was separated for 90 minutes at 102V using 0.5 μg.mL⁻¹ ethidium bromide-stained 2.5% agarose gels and visualized in a UV light apparatus (Eagle Eye II).

Results: We found 871G>A mutation in heterozygosis in three blood donors (15%), two of them typed as A3B and one as A3.

Conclusion: The 871G>A mutation is relatively commom in A3B and A3 individuals and can be easily detect by PCR-SSP.