ONE-TUBE METHOD FOR COMPLETE HPA-1 GENOTYPING BY PCR USING SEQUENCE-SPECIFIC PRIMERS

Human platelet antigens (HPA) can be targets for antibody responses th at cause life-threatening thrombocytopenia following platelet transfus ions or pregnancy As an aid to diagnosis and prevention, serologic and DNA-based methods have been developed to type HPA. Of the DNA-based s trategies, those using the polymerase chain reaction (PCR) are very se nsitive, but often require processing of amplification products. Seque nce-specific primers (SSP) in the PCR eliminate the need for extensive handling of reaction products beyond gel electrophoresis. However, cu rrent methods require a separate reaction for each allele being typed. In this report we describe a method to simultaneously and completely genotype both alleles of HPA-1 in a single PCR. In addition, because t he absence of an amplification product might also show the failure of a SSP, we introduced a recombinant template that can only be amplified by the SSP, thus ensuring primer performance and the identified genot ype.