Rapid One-Tube Long PCR-Sequencing for ABO Genotyping From Exons 2 to 7.

ABO is the most clinically important blood group system in transfusion and transplantation medicine. DNA-based methods for determining the ABO genotype have markedly advanced in recent years and have been increasingly introduced in clinical laboratories. Most studies using sequencing methods for ABO genotyping have focused on exons 6 and 7, ignoring the other exon and intron sequences. Although these approaches usually can distinguish among common ABO alleles, erroneous or ambiguous typing results might be obtained in cases of hybrid and subgroup alleles with various missense or nonsense mutations. For more accurate ABO genotype results, the entire exon sequence including the introns and regulatory regions should be analyzed, but such sequencing methods are time-consuming, labor intensive, and expensive.

To investigate the entire sequence of ABO exons 2–7 including introns 2–6 and excluding non-coding region of exon 7, long shuttle one-tube PCR-sequencing method was designed. We used a rapid DNA polymerase with high fidelity (Phusion^® Flash II DNA Polymerase; Finnzyme OY, Espoo, Finland). ABO genotyping was performed using this technique in peripheral blood of two unrelated families.

Time requirement of the PCR amplification and purification processes were about 1.8 h and 20 min, respectively. Also, this technique could read the entirety of exons 2–7 including introns, required one PCR tube, one purification step, and 27 sequencing reactions for the entire sequencing. A total of seven different ABO alleles from two families were analyzed. All genotyping results agreed with serologic findings and results expected by Mendelian inheritance. The ABO phenotypes (genotypes) of family A members were A1 (A105/A105), O (O01/O02), A1 (A105/O01), A1 (A105/O02), and A1 (A105/O02) in the husband, wife, and three sons, respectively. The ABO genotype of husband in family B was cis-ABO1/O01 and his serologic ABO typing result was ABweak. His wife had the common AB phenotype (A1B); however, her genotype revealed a hybrid of A and B alleles and the B101 allele. The hybrid allele comprised the consensus ABO allele in exons 2–5 (including introns 2–4), the B101 allele in exon 6 (with introns 5 and 6), and the Al01 allele in exon 7.

This method is suitable for the fast analysis of ABO genotypes and may be valuable in clinical transfusion or forensic applications.

No relevant conflicts of interest to declare.