FLUORESCENCE ENERGY-TRANSFER DETECTION AS A HOMOGENEOUS DNA DIAGNOSTIC METHOD

A homogeneous DNA diagnostic assay based on template-directed primer e xtension detected by fluorescence. resonance energy transfer, named te mplate-directed dye-terminator incorporation (TDI) assay, has been dev eloped for mutation detection and high throughput genome analysis, Her e, we report the successful application of the TDI assay to detect mut ations in the cystic fibrosis transmembrane conductance regulator (CFT R) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fib rosis, hemochromatosis, and multiple endocrine neoplasia, type 2, resp ectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyr ibonucleoside triphosphates before the primer extension reaction is pe rformed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a u seful clinical DNA diagnostic method.