HLA-DRB fluorotyping by dark quenching and automated analysis.

In fluorescence-based sequence-specific primed polymerase chain reaction (PCR), referred to as fluorotyping for HLA typing, a major problem is the overlap of the emission spectra of the single dyes. In order to increase the robustness of the previously described HLA-DRB1,3,4,5 low-resolution fluorotyping method, we have constructed two probes quenched by the non-fluorescent acceptor dye DABCYL. The HLA-DRB-specific probe was labeled with FAM, and the internal control probe with TAMRA, respectively, as reporter fluorescent dyes. TAMRA was replaced by DABCYL as a quencher, which led to increased robustness and better discrimination between negative and positive amplification results. ROX was used as a reference to normalize the fluorescence of the reporter dyes. Moreover, as FAM and TAMRA differ strongly by their emission maxima, HLA-DRB-specific and internal control amplification could be clearly distinguished. To further automate data analysis, the software of the TaqMan system 7700 was supplemented by an EXCEL-based calculation table, which directly took over the data. Using modified fluorotyping chemistry and automated data analysis, a total of 201 DNA samples was typed correctly. In summary, HLA-DRB fluorotyping by dark quenching and automated analysis proved to be a robust and reliable tool for research and routine purposes.