Fluorescence energy transfer detection as a homogeneous DNA diagnostic method.
A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.
Chen
X
X
Division of Dermatology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8123, St. Louis, MO 63110, USA.
Zehnbauer
B
B
Gnirke
A
A
Kwok
P Y
PY
D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D029721:Drosophila Proteins; D004735:Energy Transfer; D005453:Fluorescence; D030342:Genetic Diseases, Inborn / Q000175:diagnosis*; D005821:Genetic Techniques; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics; D000071020:Hemochromatosis Protein; D006579:Heterozygote; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D006720:Homozygote; D006801:Humans; D008565:Membrane Proteins; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics
C505032:CFTR protein, human; D029721:Drosophila Proteins; C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila