Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-02-23
2000-02-15
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 231, 536 243, 935 76, 935 77, 935 78, C12Q 168, C12P 1934, C07H 2102, C07H 2104, C12N 1500
Patent
active
060251395
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND TO THE INVENTION
This application relates to a method for the detection of mutations, including previously unknown mutations, in a gene or a gene fragment having a known wild-type sequence.
Many diseases and conditions have been found to be associated with genetic mutations, and more such associations are being identified as time goes by. In some cases, such as sickle-cell anemia, a single base change has been identified as the causative mutation. More generally, however, many different mutations may manifest themselves as a single disease. To test for each of these mutations individually using hybridization-based tests would require the development of as many different probes as there are mutations. Such an effort would involve substantial expense, however, such that in most cases hybridization-based diagnostics are only available for the most prevalent mutations. Furthermore, because development of mutation-specific hybridization-based diagnostics requires knowledge of the mutant sequence, development of diagnostic tests must await the identification and characterization of any given mutation in at least one individual.
The present invention overcomes some of the limitations of hybridization-based diagnostics by providing an assay which relies only upon knowledge of the wild-type sequence, and which detects all types of mutations, i.e., point mutations, insertions and deletions. The method involves the use of a set of oligonucleotide probes which hybridize in series along the length of the gene; the ligation of the probes together to form ligation products; and the evaluation of the sizes and quantities of the ligation products. When the gene being analyzed corresponds to the normal sequence, a characteristic pattern of ligation products is formed, including a substantial amount of full length product which results from all of the probes in the set being ligated together. When a mutation appears in the gene, the hybridization of the probe overlapping the mutation is impaired, with the result that some or all of the ligation product is of smaller size. By evaluating the sizes and quantitites of the ligation products; both the existence of a mutation and its approximate position can be identified. A definitive identification of the mutation can then be made by direct sequencing over a very restricted set of positions.
EP-A-0 185 494 discloses an assay for detection of specific nucleotide sequences in which two oligonucleotide probes which perfectly match adjacent portions of the expected sequence are hybridized and then ligated together. The assay is said to improve reliability over a plain hybridization assay, since it is unlikely that both probes would bind to a spurious location in ligatable proximity.
Ligase enzymes have previously been used in diagnostics for the detection of known point mutations. As described in Landegren et al., "A Ligase-Mediated Gene Detection Technique", Science 241: 1077-1080 (1988), and as shown in FIG. 1, this technique involved hybridization of two probes 1 and 1' to the target gene fragment 2 being analyzed. The probes 1 and 1' are selected such that the terminal base in one fragment aligns with the known mutation site when this probe is hybridized with the target gene fragment. The terminal base of the other probe aligns with the base immediately adjacent to the known mutation site. When the probes are completely complementary to the target sequence, T4 DNA ligase will couple the two probes together into a single fragment. When a mutation is present, however, the absence of hybridization at the end of one of the probes will prevent (or at least substantially reduce) the amount of ligation which occurs. By labeling one probe fragment with a capture moiety such as biotin, and the other probe fragment with a radiolabel, Landegren et al. were able to identify the presence or absence of the specific point mutation based upon formation (or non-formation) of the ligated molecule which incorporated both the capture moiety and the radiolabel.
The basic technique describ
REFERENCES:
patent: 4719180 (1988-01-01), Eaton et al.
patent: 5042272 (1991-08-01), Hartley
patent: 5132215 (1992-07-01), Jayaraman
patent: 5219734 (1993-06-01), Royer et al.
patent: 5366877 (1994-11-01), Keith
patent: 5650274 (1997-07-01), Kambara et al.
patent: 5792607 (1998-08-01), Backman et al.
patent: 5800994 (1998-09-01), Martinelli et al.
patent: 5866337 (1999-02-01), Schon
patent: 5888731 (1999-03-01), Yager et al.
Barany, F., "Genetic disease detection and DNA amplification using cloned thermostable ligase" Proc. Natl. Acad.Sci. USA vol. 88, pp. 189-193 (1991).
Grossman et al., "High-density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence-coded separation"Nucleic Acids Research vol. 22 No. 21 pp. 4527-4534 (1994).
Landegren et al., "A Ligase-Mediated Gene Detection Technique" Science vol. 241 pp.1077-1080 (1988).
Marsh et al., "Pyrococcus furious DNA Ligase and the Ligase Chain Reaction" Strategies in molecular biology vol. 5 pp. 73-76.
Rouwendal et al., "Simultaneous Mutagenesis of Multiple Sites: Application of the Ligase Chain Reaction Using PCR Products Instead of Oligonucleotides" Circle Reader Service No. 172 vol. 15 pp. 68-75 (1993).
Wu et al., "Specificity of the nick-closing activity of bacteriophage T4 DNA ligase" Gene 76 pp. 245-254 (1989).
Weisberg et al., "Simultaneous Mutagenesis of Multiple Sites: Appication of the Ligase Chain Reaction Using PCR Products Instead of Oligonucleotide", Biotechnique15: 68-76 (1993).
Nickerson et al., "Automated DNA Diagnostics Using an ELISA-based Oligonucleotide Ligation Assay", Proc. Nat'l Acad. Sci. (USA) 87: 8923-8927 (1990).
Dunn James M.
Yager Thomas D.
Jones W. Gary
Visible Genetics Inc.
Whisenant Ethan
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