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.
Slateva
K
K
Department of Transfusion Medicine, Hannover Medical School, Hannover, Germany.
Elsner
H A
HA
Albis-Camps
M
M
Blasczyk
R
R
D001330:Electronic Data Processing; D005456:Fluorescent Dyes; D006680:HLA Antigens / Q000032:analysis / Q000145:classification*; D006684:HLA-DR Antigens / Q000235:genetics; D059811:HLA-DRB1 Chains; D059826:HLA-DRB3 Chains; D059845:HLA-DRB4 Chains; D059847:HLA-DRB5 Chains; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012235:Rhodamines / Q000737:chemistry; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000737:chemistry
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005456:Fluorescent Dyes; D006680:HLA Antigens; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D059826:HLA-DRB3 Chains; D059845:HLA-DRB4 Chains; D059847:HLA-DRB5 Chains; D012235:Rhodamines; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene