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.

[1]  D. Clayton,et al.  Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC , 1994, Nature Genetics.

[2]  Alfred A. Boyd,et al.  Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2 , 1996, Nature Genetics.

[3]  K. Livak,et al.  Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. , 1995, PCR methods and applications.

[4]  P. Goodfellow,et al.  Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  James L. Winkler,et al.  Accessing Genetic Information with High-Density DNA Arrays , 1996, Science.

[6]  M. Hanke,et al.  Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. , 1994, BioTechniques.

[7]  B. Ponder,et al.  Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A , 1993, Nature.

[8]  M. C. Ellis,et al.  A novel MHC class I–like gene is mutated in patients with hereditary haemochromatosis , 1996, Nature Genetics.

[9]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[10]  N Risch,et al.  The Future of Genetic Studies of Complex Human Diseases , 1996, Science.

[11]  R. Hofstra,et al.  A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma , 1994, Nature.

[12]  S. P. Fodor,et al.  Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two–colour fluorescence analysis , 1996, Nature Genetics.

[13]  F. Couch,et al.  Mutations and Polymorphisms in the familial early‐onset breast cancer (BRCA1) gene , 1996, Human mutation.

[14]  L. Tsui,et al.  Erratum: Identification of the Cystic Fibrosis Gene: Genetic Analysis , 1989, Science.

[15]  S. Wells,et al.  Predictive testing for multiple endocrine neoplasia type 2A (MEN 2A) based on the detection of mutations in the RET protooncogene. , 1994, Surgery.

[16]  F. Collins,et al.  The carrier frequency of the BRCA1 185delAG mutation is approximately 1 percent in Ashkenazi Jewish individuals , 1995, Nature Genetics.

[17]  P. Kwok,et al.  Template-directed dye-terminator incorporation (TDI) assay: a homogeneous DNA diagnostic method based on fluorescence resonance energy transfer. , 1997, Nucleic acids research.

[18]  P. Goodfellow,et al.  Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. , 1993, Human molecular genetics.

[19]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.