A modified PCR‐SSP method for the identification of ABO blood group antigens

Abstract

The ABO blood group antigens are carbohydrate molecules synthesized by the glycosyltransferases encoded by the ABO gene on chromosome 9. Kidney transplantation across the ABO barrier generally leads to rapid humoral graft rejection due to the presence of naturally occurring antibodies to the A and B antigens. We have developed a method for ABO typing our cadaveric organ donors by the polymerase chain reaction using sequence-specific primers (PCR-SSP). The method uses 12 primers in eight PCR mixtures and is performed under the same conditions as our routine HLA-A, B, C PCR-SSP typing. The PCR-SSP-based types of 166 regular blood donors and 148 cadaveric organ donors all showed total concordance with their serologically assigned ABO groups. Six individuals possessing the ABO A subgroups (A3, Ax and Aend) all typed as A1 by PCR-SSP, as expected. PCR-SSP is an appropriate method for ABO typing of cadaveric organ donors and, importantly, enables both ABO and HLA typing to be performed on the same DNA material.

A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

“…Yamamoto and his colleagues reported the molecular genetic basis of the ABO group in 1990 [2], permitting analysis of ABO blood groups at the DNA level. Therefore, various PCR-based methods for ABO genotyping have been developed, including PCR-RFLP [3], MS-PCR [4], multiplexed allele-specific PCR [5,6], PCR-SSP [7,8], the universal reporter primer system [9], and real-time PCR using allele-specific primers [10]; these methods have been shown to be useful in forensic investigations. However, most of these methods have shortcomings in terms of the need for post-PCR manipulations, such as enzyme digestion and electrophoresis, and these steps can result in errors and carry-over contamination.…”

“…PCR-RFLP is the most well-known method for ABO genotyping; however, incomplete restriction enzyme digestion and simultaneous use of restriction enzymes can result in mistyping [10,21]. PCR-SSP requires size separation of PCR fragments by gel electrophoresis and pseudopositive signals from unexpected primer extension reactions often lead to errors in ABO genotyping [8,21,22]. The allele-specific PCR method enables the simultaneous detection of SNP sites by multiplex PCR.…”

“…Since Yamamoto and colleagues reported the differences in the nucleotide sequences of ABO alleles [1,2], numerous methods for ABO genotyping based on polymerase chain reaction (PCR) have been developed, such as PCR-restriction fragment length polymorphism (PCR-RFLP) analysis [3], mutagenically separated PCR (MS-PCR) [4], multiplexed allele-specific PCR [5,6], PCR with sequence-specific primers (PCR-SSP) [7,8], universal reporter primer systems [9], and real-time PCR using allele-specific primers [10]. However, most of these methods require multiple post-PCR manipulation steps, including electrophoresis and enzyme digestion; for this reason, these methods often have a risk of carry-over contamination [10].…”

ABO genotyping in leukemia patients reveals new ABO variant alleles

“…Critical mutations for A and B alleles have been found predominantly in exons 6 and 7 of the ABO gene, where the catalytic domain of ABO glycosyltransferases is encoded (Olsson and Chester, 2001;Downing and Darke, 2003). In this study, we typed for ABO and genotyped exons 6 and 7 of the ABO gene, using PCR-ASP and DNA sequencing, in 108 patients with leukemia to identify variations and polymorphisms.…”

Molecular characterisation of the monocytic cell line THP‐1 demonstrates a discrepancy with the documented HLA type

“…Both of these in-house genotyping systems are based on methods which have been previously published. 3,4 Using this assay, we identified the following HLA type for THP-1. HLA-A*02; B*15; C*03; DRB1*01, DRB1*15; DRB5*01/02; DQB1*05 and DQB1*06 (and ABO blood group genotype BB).…”