Novel polymorphisms in exons 6 and 7 of A/B alleles detected by polymerase chain reaction-single strand conformation polymorphism

Abstract

Background and objectives: The ABO blood group system is the most important blood group system in transfusion medicine. In addition to the major A, B and O alleles, many rare alleles have been defined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and analysis by PCR sequence specific primers (SSP) are commonly conducted for genotyping but have the limitation of being unable to detect unknown substitution(s) in amplified DNA fragments, whereas PCR-single strand conformation polymorphism (SSCP) can be used for both.

Materials and methods: Three-hundred unrelated blood donors of the AB phenotype were enrolled. Four pairs of primers were designed to constitute two sets of multiplex PCRs: this amplifies four fragments spanning the entire exon 6 and its immediate flanking regions, nucleotides 432-1065, as well as the 3' untranslated region of exon 7 of the ABO gene. The SSCP electrophoresis was carried out on a 12.5% polyacrylamide gel in a GenePhor electrophoresis unit. For those with unexpected banding patterns, SSCP analyses were performed in duplicate and samples were cloned and sequenced for exons 6 and 7.

Results: Seven samples were noted to have six variant alleles, of which five have not been previously reported in the literature. Of these five novel variants, four were derived from the B allele, while the other derived from the A allele.

Conclusions: By using PCR-SSCP, five novel A/B alleles were found.