Similarly, the sequences around the first and second of the four amino acid substitutions that discriminate A/O and B alleles can be cleaved by BssHII and NarI, and HpaII and AluI, respectively. Using restriction fragment length polymorphism (RFLP), we confirmed the presence of these differences in genomic DNAs from the cell lines used to prepare cDNA libraries (these distinctions in RNAs were previously determined by the nucleotide sequencing of cloned cDNAs). As opposed to KpnI and BstEII, which recognize and cleave longer nucleotide sequences, HpaII and AluI are frequent cutters, recognizing and cleaving shorter sequences. As such, we used PCR to amplify DNA fragments containing those restriction sites and then digested with these enzymes, rather than Southern hybridization. We used the same strategy for the BssHII and NarI distinction, though they are not frequent cutters because of easier experimental protocols. When we performed these experiments, the specificity of the Taq DNA polymerase was not as good as it is now. Therefore, several additional bands are visible in the background. Nonetheless, the presence/absence of the restriction sites could be determined easily.