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

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Abstract
The aim of our study was to develop a fluorotyping strategy for the HLA‐A locus. In contrast to conventional sequence‐specific primed PCR (PCR‐SSP), which is based on an agarose gel electrophoresis, fluorotyping eliminates the drawback of low sample throughput, low potential for automation and problems related to contamination. Additionally, fluorotyping is capable of delivering quantitative results depending on the system set‐up. The fluorescence‐based PCR‐SSP assay relies on target‐specific and individually labeled fluorogenic 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‐carboxyfluorescein (FAM) as the reporter and at its 3′ end with 6‐carboxy‐tetramethylrhodamine (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 Taq DNA‐polymerase 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 individual PCR primer mixes. The internal control amplicon was detected with a tetrachloro‐6‐carboxyfluorescein‐TAMRA‐labeled fluorogenic probe. Since the HLA‐A amplicons had to include the 2nd intron as the target for the fluorogenic probe, the sequence motifs which could be used as priming sites were limited. Therefore, some primer mixes covered more than one specificity resulting in ambiguous amplification patterns in 31 of 231 possible allele or group combinations of HLA‐A1‐A80. These ambiguities, which all involved the inability to discriminate a particular heterozygous genotype from a homozygous genotype, may be resolved by the quantitative data revealed by fluorotyping. This feature is also helpful to detect new alleles which are not amplified by the current primer mixes.