ABO genotyping by inverse PCR technique

Inverse PCR technique was applied to type three major alleles (A(1), B and O(1)) of the ABO blood group by simultaneously detecting separated allele-determining nucleotides (the 261st base in exon 6 and the 796th and 803rd nucleotides in exon 7) of the ABO gene. A sequence of about 1.7 kb from exons 6 to 7 of each allele was amplified, both termini of the fragment ligated, and allele-typing performed by the inverse PCR-restriction fragment length polymorphism (IP-RFLP) and allele-specific inverse-PCR (ASIP) methods. For intramolecular ligation, primers for the first PCR were designed to have Acc I-restriction sites within the sequences, and both termini of the 1.7-kb fragment were digested with Acc I. Using the IP-RFLP method, the inverse PCR product was digested with Kpn I, Nla III and Dde I, A(1), B, O(1)-standard (O(A)) and O(1)-variant (O(G)) alleles were detected as 365-bp, 272-bp, 193-bp and 128-bp fragments, respectively. By the ASIP method using four allele-specific primers, 222-bp, 124-bp and 232-bp fragments were amplified from A(1), B and O(1) templates, respectively. These techniques would be applicable to detecting separated polymorphic regions of some other genes.