Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-14
2001-03-13
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06200752
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is in the field of detecting the presence of a nucleic acid sequence in a sample.
2. Description of the Background Art
Fluorescence in situ hybridization (“FISH”) is widely used in medical diagnostics. Present technology for in situ hybridization includes the use of fluorescence probes such as fluorescein and rhodamine and detection of hybridized DNA by localization of the product of an enzyme catalyzed reaction.
The following references describe known fluorescence in situ hybridization techniques:
Swiger, R. R., and Tucker, J. D., “Fluorescence In Situ Hybridization: A Brief Review,”
Environmental and Molecular Mutagenesis
27:245-54 (1996).
Schrock, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson-Smith, M. A., Ning, Y., Dedbetter, D. H., Bar-Am, I., Soenksen, D., Garini, Y., and Ried, T., “Multicolor Spectral Karyotyping of Human Chromosomes,”
Science
273:494-98 (1996).
Lewis, R., “Chromosome Charting Takes a Giant Step,”
Photonics Spectra
48-50 (1996).
Fox, J. L., Hsu, P., Legator, M., Morrison, L. E., and Seelig, S. A., “Fluorescence In Situ Hybridization: Powerful Molecular Tool for Cancer Prognosis,”
Clinical Chemistry
41:1554-59 (1995).
Nederlof, P. M., van der Flier, S., Wiegant, J., Raap, A. K., Tanke, H. J., Ploem, J. S., and van der Ploeg, M., “Multiple Fluorescence In Situ Hybridization,”
Cytometry
11:126-31 (1990).
Siadat-Pajouh, M., Ayscue, A. H., Periasamy, A., and Herman, B., Introduction of a Fast and Sensitive Fluorescent In Situ Hybridization Method for Single-copy Detection of Human Papillomavirus (HPV) Genome,”
The Journal of Histochemistry and Cytochemistry
42:1503-12 (1994).
Kearns, W. G. and Pearson, P. L., “Fluorescent In Situ Hybridization Using Chromosome-Specific DNA Libraries,”
Methods in Molecular Biology
33:15-22 (1994).
Tkachuk, D. C., Pinkel, D., Kuo, W., Weier, H., and Gray, J. W., “Clinical Applications of Fluorescence in situ Hybridization,”
GATA
8(2):67-74 (1991).
Denijn, M., Schuurman, H., Jacobse, K. C., and Weger, R. A., “In Situ hybridization: A valuable tool in diagnostic pathology,”
APMIS
100:669-681 (1992).
McNeil, J. A., Villnave Johnson, C., Carter, K. C., Singer, R. H., and Bentley Lawrence, J., “In Situ Hybridization,”
GATA
8(2):41-58 (1991).
Schwarzachter, T., and Heslop-Harrison, J. S., “Direct Fluorochrome-Labeled DNA Probes for Direct Fluorescent In Situ Hybridization of Chromosomes,”
Methods in Molecular Biology, Vol.
28:
Protocols for Nucleic Acid Analysis by Nonradioactive Proves
167-76 (1994).
Durrant, J., Brunning, S., Eccleston, L., Chadwick, P., and Cunningham, M., “Fluorescein as a label for non-radioactive in situ hybridization,”
Histochemical Journal
27:94-99 (1995).
None of these papers mentions the use of metal-ligand complexes in fluorescence in situ hybridization to detect the presence of a nucleic acid.
One limitation of prior art DNA hybridization is the low levels of light available from commonly used fluorophores, the presence of significant background fluorescence which limits sensitivity, and photobleaching of the probes. The resulting fluorescence is typically on a nanosecond time scale, which is also the decay time of the commonly used fluorophores. In addition, the commonly used fluorophores display small Stoke's shifts making it difficult to detect their fluorescence in the presence of a fluorescent background. There is also a need for fluorophores with greater resistance to fading and increased shelf life, i.e., archivability of the slides.
There is extensive literature regarding the spectral properties of metal-ligand complexes. The following is a list of papers regarding metal-ligand complexes:
Maestri, M., Sandrini, D., Balzani, V., Maeder, U. and von Zelewsky, “Absorption Spectra, Electrochemical Behavior, Luminescence Spectra, and Excited-State Lifetimes of Mixed-ligand Ortho-Metalated Rhodium(III) Complexes,”
Inorg. Chem.,
26:1323-1327 (1987).
Sutin, N. and Creutz, C., “Properties and Reactivities of the Luminescent Excited States of Polypyridine Complexes of Ruthenium(II) and Osmium(II),”
Inorg.
&
Organometall. Photochem.,
Chap. 1, pp. 1-27 (1978).
Hager, G. D., Watts, R. J. and Crosby, G. A., “Charge-transfer Excited States of Ruthenium(II) Complexes. Relationship of Level Parameters to Molecular Structure,”
J. Am. Chem. Soc.,
97;7037-7042 (1975).
Orellana, G. and Braun, A. M., “Quantum Yields of
3
MLCT Excited State Formation and Triplet-Triplet Absorption Spectra of Ruthenium(II) Tris-Chelate Complexes Containing Five- and Six-Membered Heterocyclic Moieties,”
J. Photochem. Photobiol. A. Chem..,
48:277-289 (1989).
Harrigan, R. W. and Crosby, G. A., “Symmetry Assignments of the Lowest CT Excited States of Ruthenium(II) Complexes Via a Proposed Electronic Coupling Model,”
J. Chem. Phys.,
59(7):3468-3476 (1973).
Yersin, H. and Braun, D., “Isotope-Induced Shifts of Electronic Transitions: Application to [Ru(bpy-h
8
)
3
]
2+
and [Ru(bpy-d
8
)
3
]
2+
in [Zn(bpy-h
8
)
3
] (ClO
4
)
2
,” Chem. Phys. Letts.,
179(1,2):85-94 (1991).
Coe, B. J., Thompson, D. W., Culbertson, C. T., Schoonover, J. R. and Meyer, T. J., “Synthesis and Photophysical Properties of Mono(2,2′,2′-Terpyridine) Complexes of Ruthenium(II),”
Inorg. Chem.,
34:3385-3395 (1995).
Lees, A. J., “Luminescence Properties of Organometallic Complexes,”
Chem. Rev.,
87:711-743 (1987).
DeArmond, M. K. and Carlin, C. M., “Multiple State Emission and Related Phenomena in Transition Metal Complexes,”
Coordination Chem. Rev.,
36:325-355 (1981).
Kondo, T., Yanagisawa, M. and Fujihira, M., “Single Exponential Decay for the Luminescence Intensity of Ru(bpy)
3
2+
Complex in Langmuir-Blodgett Films,”
Chem. Letts.,
1639-1993 (1993).
None of the above references suggest use of metal-ligand complexes in fluorescence in situ hybridization.
There remains a need in the art for improved methods of detecting the presence of nucleic acid sequences.
SUMMARY OF THE INVENTION
In accordance with the present invention, a method for detecting the presence of a nucleic acid sequence in a sample includes the step of coupling a fluorescent metal-ligand complex to an oligonucleotide having a sequence, complementary to the nucleic acid sequence to form a probe. The probe is added to a sample that contains the nucleic acid sequence to form a mixture containing a reaction product. The mixture is exposed to an exciting amount of radiation. The fluorescence of the metal-ligand complex is detected, and the presence of the nucleic acid sequence is determined based on the fluorescence of the metal-ligand complex.
Also in accordance with the present invention a composition for detecting the presence of a nucleic acid sequence includes a fluorescent metal-ligand complex coupled to an oligonucleotide having a sequence complementary to the nucleic acid sequence.
Further in accordance with the present invention a diagnostic kit for detecting the presence of a nucleic acid sequence includes a fluorescent metal-ligand complex coupled to an oligonucleotide having a sequence complementary to the nucleic acid sequence.
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Denijn et al., In Situ Hybridization : A Valuable Tool in Diagnostic Pathology. AMPIS 100 : 669-681 (1992).
Collins et al., J. of Forensic Sciences 39(6) : 1347-1355 (1994).
Guo et al., “A Long-Lived, Highly Luminescent Re (I) Metal Ligand complex as a Biomolecular Probe”, Analytical Biochemistry 254 : 179-186 (1997).
Collins et al., “Identification of Sperm and Non-sperm Male Cells in Cervicovaginal Smears Using Fluorescen
Rothwell Figg Ernst & Manbeck
Whisenant Ethan
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