Fluorotyping of HLA-A by sequence specific priming and fluorogenic probing

The aim of our study was to develop a fluorotyping strategy for the HLA-A l ocus. In contrast to conventional sequence-specific primed PCR (PCR-SSP), w hich is based on an agarose gel electrophoresis, fluorotyping eliminates th e drawback of low sample throughput, low potential for automation and probl ems related to contamination. Additionally, fluorotyping is capable of deli vering quantitative results depending on the system set-up. The fluorescenc e-based PCR-SSP assay relies on target-specific and individually labeled fl uorogenic probes allowing a simultaneous and differential detection of the specific HLA and the internal control product. The probe used to detect the HLA-A specific amplicons was labeled at its 5' end with 6-carboxyfluoresce in (FAM) as the reporter and at its 3' end with 6-carboxy-tetramethylrhodam ine (TAMRA) as the quencher. The probe hybridized within the 2nd intron to a conserved region which was found to be identical in all HLA-A alleles and was covered by all primer mixes. During successful PCR the cleavage of the FAM-labeled probe through the 5'-3' exonuclease activity of the Tag DNA-po lymerase led to an interruption of the TAMRA-mediated quenching effect and generated a significant increase of the FAM fluorescence. The specific HLA- A typing information was based on the FAM fluorescence released by 24 indiv idual PCR primer mixes. The internal control amplicon was detected with a t etrachloro-6-carboxyfluorescein-TAMRA-labeled fluorogenic probe. Since the HLA-A amplicons had to include the 2nd intron as the target for the fluorog enic probe, the sequence motifs which could be used as priming sites were l imited Therefore, some primer mixes covered more than one specificity resul ting in ambiguous amplification patterns in 31 of 231 possible allele or gr oup combinations of HLA-A1-A80. These ambiguities, which all involved the i nability to discriminate a particular heterozygous genotype from a homozygo us genotype, may be resolved by the quantitative data revealed by fluorotyp ing. This feature is also helpful to detect new alleles which are not ampli fied by the current printer mixes.