A homogeneous, ligase-mediated DNA diagnostic test.

Single-nucleotide variations are the most widely distributed genetic markers in the human genome. A subset of these variations, the substitution mutations, are responsible for most genetic disorders. As single nucleotide polymorphism (SNP) markers are being developed for molecular diagnosis of genetic disorders and large-scale population studies for genetic analysis of complex traits, a simple, sensitive, and specific test for single nucleotide changes is highly desirable. In this report we describe the development of a homogeneous DNA detection method that requires no further manipulations after the initial reaction is set up. This assay, named dye-labeled oligonucleotide ligation (DOL), combines the PCR and the oligonucleotide ligation reaction in a two-stage thermal cycling sequence with fluorescence resonance energy transfer (FRET) detection monitored in real time. Because FRET occurs only when the donor and acceptor dyes are in close proximity, one can infer the genotype or mutational status of a DNA sample by monitoring the specific ligation of dye-labeled oligonucleotide probes. We have successfully applied the DOL assay to genotype 10 SNPs or mutations. By designing the PCR primers and ligation probes in a consistent manner, multiple assays can be done under the same thermal cycling conditions. The standardized design and execution of the DOL assay means that it can be automated for high-throughput genotyping in large-scale population studies.

[1]  Fred Russell Kramer,et al.  Multicolor molecular beacons for allele discrimination , 1998, Nature Biotechnology.

[2]  Francis S. Collins,et al.  Variations on a Theme: Cataloging Human DNA Sequence Variation , 1997, Science.

[3]  X Chen,et al.  Fluorescence energy transfer detection as a homogeneous DNA diagnostic method. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[4]  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.

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

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

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

[8]  F. Eggerding.  A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing. , 1995, PCR methods and applications.

[9]  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.

[10]  John A. Todd,et al.  Towards fully automated genome–wide polymorphism screening , 1995, Nature Genetics.

[11]  A. Jeffreys,et al.  Allele-specific MVR-PCR analysis at minisatellite D1S8. , 1993, Human molecular genetics.

[12]  S. Gandrille,et al.  Polymorphism in the protein C gene detected by denaturing gradient gel electrophoresis. , 1991, Nucleic acids research.

[13]  P. Humphries,et al.  A sequence polymorphism in the human peripherin/RDS gene. , 1991, Nucleic acids research.

[14]  W. Weber,et al.  Diverse point mutations in the human gene for polymorphic N-acetyltransferase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

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

[16]  L. Tsui,et al.  Identification of the cystic fibrosis gene: genetic analysis. , 1989, Science.

[17]  A. Szabo,et al.  Modern quantum chemistry , 1982 .

[18]  Y. Kan,et al.  beta zero thalassemia in Sardinia is caused by a nonsense mutation. , 1981, The Journal of clinical investigation.