ABO Genotyping by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

Abstract

Background: The ABO phenotype is controlled by A, B and O genes. The B allele differs from the A allele at positons 526
(CYG), 703(G YA), 796(C -A) and 803(G-)C) which cause amino acid changes. The O allele differs from the A and B alleles at position 261(deletion of G), this causes a shift in the reading frame that creates a premature stop codon. Objective: To determine ABO genotype by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Materials and Methods: Blood samples were collected from 70 blood donors including 20 donors with group O, 20 with group A, 20 with group B and 10 with group AB. ABO phenotype was identified by red cell and serum grouping. Red cell typing was performed by standard tube tests using anti-A, anti-A1, anti-B and anti-A,B monoclonal antibodies. Serum grouping was done with standard A, and B cells. DNA was extracted from EDTA blood by the Chelex method. PCR was performed with primers mo-57/mo-46 and ABO F624-649/R770-798 to produce DNA fragments from exon 6 and 7, respectively. Exon 6 fragments from mo-57/mo-46 were digested with Kpn I which cleaved a specific GGTACC stretch in the O allele. In the exon 7 fragments from ABO F624-649/R770-798 amplicon, Hpa II cut a CCGG and Alu I cut a AGCT sequence in B alleles, respectively. Cleavage products were analyzed on 8.5% polyacrylamide gels. The polymerase chain reaction-sequence specific primer (PCR-SSP) was performed with primers 261X/261C. PCR products were electrophoresed in 2% agarose gel. Results: For the O allele, Kpn I generated 87 and 164 bp fragments for the mo-57/46 amplicons. A or B alleles lack a restriction site for Kpn I, the 252 bp amplicons were not cleaved. The 175 bp long ABO F624-649/R770-798 of A or O alleles were digested by Hpall to give 78, 19, 24 and 54 bp fragments. B alleles however gave 97, 24 and 54 bp fragments due to a base substitution at position 703(G -A). The 19, 24 and 54 long fragments cannot be detected because of the small amount of DNA in the corresponding bands. Alu I split the 175 bp ABO F624-649/R770-798 amplicons into two fragments of 81 and 94 bp for the B allele; A or O alleles were not cleaved. The ABO genotypes were determined by the intersection of the cleavage fragments from Kpn
I, Hpa I and/or Alu I as follows; 1) genotype OO : 87 and 164 bp from Kpn I; 78 bp from Hpa II; 175 bp from Alu I. 2) genotype AA : 252 bp from Kpn I; 78 bp from Hpa I; 175 bp from Alu I. 3) genotype BB: 252 bp from Kpn I; 97 bp from Hpa II; 81 and 94 bp from Alu I. 4) genotype AB: 252 bp from Kpn I; 78 and 97 bp from Hpa II; 81, 94 and 175 bp from Alu I. 5) genotype AO: 87, 164 and 252 bp from Kpn I; 78 bp from Hpa I; 175 bp from Alu I 6) genotype BO: 87, 164 and 252 bp from Kpn I; 78 and 97 bp from Hpa II; 81, 94 and 175 bp from Alu I. In the PCR-SSP, the 195 bp amplicon for O allele was found. The ABO genotypes could be evaluated using O allele detection by PCR-SSP and ABO blood grouping by serologic method. The ABO phenotypes of 70 donors were as follows: 20 O; 20 A; 18 B; 9 AB; 2 B and 1 AB. ABO genotypes by PCR-RFLP weak and PCR-SSP were as follows: 20 OO; 3 AA; 17 AO; 6 BB; 14 BO, 10 AB. Both of the B were heterozygous BO and the weak AweakB was genotype AB. Conclusion : The ABO genotype using PCR-RELP can 1) give the result concordant with PCR- SSP for O allele and combine with blood group serology 2) show the result compatible to ABO phenotype. 3) apply with DNA samples from other types of tissues.