FLUOROTYPING OF HLA-C - DIFFERENTIAL DETECTION OF AMPLICONS BY SEQUENCE-SPECIFIC PRIMING AND FLUOROGENIC PROBING

Conventional PCR-SSP, which is based on an agarose gel-based read-out, has the disadvantages of time-consuming post-PCR steps and low potent ial for automation. The aim of our study was to sort out these drawbac ks by establishing a fluorescence-based PCR-SSP system for HLA-C. The assay relies on the sequence-specific identification of amplicons with individually labeled probes that are cleaved during successful PCR by the 5'-3' exonuclease activity of the Taq-DNA Polymerase. The oligonu cleotides are labeled with a unique and spectrally resolvable fluoresc ent reporter dye at the 5' terminus (FAM or TET) and a common quencher dye at the 3' terminus (TAMRA). In case of amplification, the reporte r escapes from the quenching control caused by the physical separation of the dyes, resulting in a significant increase of the reporter fluo rescence. This allows simultaneous and differential detection of the s pecific HLA (FAM) and internal control (TET) product. The HLA-C fluoro typing information is based on the individual reporter fluorescence re leased by 18 PCR primer mixes. Using this method, we analyzed 145 samp les previously typed with conventional PCR-SSP and found a concordance rate of 100%. Furthermore, fluorotyping revealed quantitative results that may indicate the presence of homozygosity by high signal intensi ties. This provided extra protection not to miss new alleles which are not amplified by the current primer mixes. These features as well as the capability of high sample throughput and the possibility of automa tion makes fluorotyping an attractive tool for PCR-based HLA typing.