Human platelet antigen‐2 and ‐3 genotyping by PCR‐SSP

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Abstract
SUMMARY. Allele-specific PCR using sequence specific primers (PCR-SSP) is a simple and reliable technique to detect point mutations in genes. We have developed a PCR-SSP to enable the detection of a C-T mutation at position 482 of the GPIb gene and a T-G mutation at position 13962 in exon 26 of the GPIIb gene. These point mutations are at the basis of the HPA alloantigens 2a, 2b and 3a, 3b respectively. One primer of each primer set has a 3′ nucleotide complementary to the DNA sequence coding for one allele. PCR product is only produced when the corresponding DNA is present and thus the genotype is determined by the presence or absence of a band in agarose electrophoresis of PCR products. A second set of primers in the same reaction yields a product regardless of the HPA genotype to control the efficiency of the PCR amplification. The HPA-2 and -3 genotypes determined in this way were in strict concordance with those established by conventional genotyping using PCR followed by restriction enzyme digestion (PCR-ASRA). PCR-SSP is a rapid and reliable technique that can be used for the determination of alleles which code for platelet alloantigens.

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