Before our elucidation of the molecular genetic basis of the human ABO blood group system in 1990, it was impossible to discriminate RBCs from individuals with an AA genotype from RBCs taken from individuals with an AO genotype. The determination of the nucleotide sequences of the ABO alleles and the identification of a single nucleotide polymorphism among those alleles allowed us to genotype the ABO genetic locus. We used two different methods in the original study. We used the restriction fragment length polymorphism (RFLP) followed by Southern hybridization with the human A transferase probe to discriminate both the alleles with the O allele-specific single nucleotide deletion (to be cleaved with KpnI) and the alleles without the deletion (to be cleaved with BstEII). We used PCR to amplify the DNA fragments that contained the differences in the nucleotide sequence between the A/O and B alleles. The amplified DNA fragments were later subjected to allele-specific restriction enzyme digestion (BssHII and AluI for the A/O alleles and NarI and HpaII for the B allele). We later performed successful allele-specific PCR using fluorescence-labeled primers. However, we decided not to publish the results because Hood and colleagues reported the use of LCR (ligase chain reaction) to discriminate ABO alleles using fluorescence detection. Later, a variety of methods were also applied to ABO genotyping to detect the single nucleotide polymorphism.
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