A new method for ABO genotyping using a multiplex single-base primer extension reaction and its application to forensic casework samples.

We developed a new method for ABO genotyping using a multiplex single-base primer extension reaction. The method allows for the simultaneous detection of six SNP sites in the ABO gene (nt 261, 297, 681, 703, 802, and 803) and the determination of ABO genotypes from their combinations. It enabled ABO genotyping of all samples of peripheral blood DNA extracted from 103 Japanese individuals, and had a highly satisfactory detection sensitivity being capable of genotyping 0.1 ng of genomic DNA. Using this method, we were able to determine ABO genotypes of minute stain samples, heated bloodstains, aged bloodstains and mixed samples. Experiments with samples from 26 animal species and bacterial samples to test the species-specificity of the method showed that genotyping was possible in the chimpanzee and gorilla, but their genotypes were extremely rare in humans. In addition, we applied this method to casework samples, and successfully determined ABO genotypes of bones, teeth, muscles, organs, nails, and semen-contaminated vaginal fluid in which ABO grouping by conventional serological techniques was not possible. This new method enables the sensitive, simultaneous detection of six SNP sites in the ABO gene by two specific reactions, i.e. PCR and a primer extension reaction. Therefore, it holds promise as an effective method of ABO genotyping particularly for forensic samples.