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HPA‐1, ‐2 and ‐3 genotyping using the TaqMan™ reaction
Author(s) -
Hurd C.,
Ocheja A.,
CheethamWilkinson C.,
Ouwehand W.
Publication year - 2000
Publication title -
transfusion medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.471
H-Index - 59
eISSN - 1365-3148
pISSN - 0958-7578
DOI - 10.1046/j.1365-3148.2000.00261-6.x
Subject(s) - taqman , genotyping , ethidium bromide , microbiology and biotechnology , taq polymerase , genotype , biology , polymerase chain reaction , genomic dna , agarose , allele , genetics , dna , gene , thermus aquaticus
Human platelet alloantigen (HPA) genotyping is widely used (i) in the diagnosis and management of the alloimmune thrombocytopenias; (ii) in the HPA typing of donor panels for the provision of HPA‐matched blood products; and (iii) in the study of HPA and cardiovascular disease association. We describe HPA‐1, ‐2 and ‐3 genotyping by allelic discrimination using the Taqman™ reaction. The assay combines the PCR reaction, using platelet glycoprotein‐specific primers, with the release of a reporter fluorophore from an allele‐specific oligonucleotide probe by the 5′−3′ exonuclease activity of Taq polymerase. By using two differently labelled allele‐specific probes and in situ measurement of released fluorescence, the genotype of each biallelic system can be determined in a single PCR reaction without additional post‐PCR manipulation. The TaqMan™ assay has been compared with the PCR using allele specific primers (PCR‐SSP) evaluated by ethidium bromide staining in agarose gels. Parallel testing of 253 genomic DNA samples showed that the TaqMan™ assay to be more consistent and to be independent of subjective interpretation errors. Subjective reading of weak bands accounted for the misinterpretation of the PCR‐SSP evaluated on ethidium bromide gels. The TaqMan™ reaction is readily adaptable to high throughput genotyping by automation of the dispensing and data handling steps.