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

Abstract

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.