Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-15
2002-08-13
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091100, C435S091200, C252S400610, C252S519130, C436S546000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06432637
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is in the field of determining a base sequence of a nucleotide strand.
2. Description of the Background Art
The field of DNA sequencing is very active because of the decision to sequence the human genome. Presently available technology for determining a base sequence of a nucleotide strand uses different fluorescence labels on the four nucleotides, adenine, thymine, guanine, and cytosine, during sequencing. The nucleotide is identified by the emission spectrum which is distinct for each of the four probes used for each nucleotide.
The following references describe known DNA sequencing techniques which utilize measurement of fluorescence intensity:
T. Hunkapiller, R. J. Kaiser, B. F. Koop, and L. Hood, “Large-Scale and Automated DNA Sequence Determination,”
Science
254:59-67 (1991).
D. B. Shealy, M. Lipowska, J. Lipowski, N. Narayanan, S. Sutter, L. Strekowski, and G. Patonay, “Synthesis, Chromatographic Separation, and Characterization of Near-Infrared-Labeled DNA Oligomers for Use in DNA Sequencing,”
Analytical Chemistry
67:247-251 (1995).
J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer, and R. A. Mathies, “Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis,”
Biophysics
92:4347-51 (1995).
J. Ju, A. N. Glazer, and R. A. Mathies, “Energy transfer primers: A new fluorescence labeling paradigm for DNA sequencing and analysis,”
Nature Medicine
2:246-49 (1996).
L. M. Smith, J. Z. Sanders, R. J. Kaiser, P. Hughes, C. Dodd, C. R. Connell, C. Heiner, S. B. H. Kent, and L. E. Hood, “Fluorescence detection in automated DNA sequence analysis,”
Nature
321:674-79 (1986).
D. C. Williams and S. A. Soper, “Ultrasensitive Near-IR Fluorescence Detection for Capillary Gel Electrophoresis and DNA Sequencing Applications,”
Analytical Chemistry,
67:3427-32.
S. Wiemann, J. Stegemann, D. Grothues, A. Bosch, X. Estivill, C. Schwager, J. Zimmermann, H. Voss, and W. Ansorge, “Simultaneous On-Line DNA Sequencing on Both Strands with Two Fluorescent Dyes,”
Analytical Biochemistry
224:117-21 (1995).
K. C. Huang, M. A. Quesada, and R. A. Mathies, “DNA Sequencing Using Capillary Array Electrophoresis,”
Anal. Chem.
64:2149-54 (1992).
J. M. Prober, G. L. Trainor, R. J. Dam, F. W. Hobbs, C. W. Robertson, R. J. Zagursky, A. J. Cocuzza, M. A. Jensen, and K. Baumeister, “A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides,”
Science
238:336-41 (1987).
S. Takahashi, K. Murakami, T. Anazawa, and H. Kambara, “Multiple Sheath-Flow Gel Capillary-Array Electrophoresis for Multicolor Fluorescent DNA Detection,”
Anal. Chem.
66:1021-26 (1994).
The following references describe known DNA sequencing techniques which utilize measurement of fluorescence lifetime:
M. Sauer, K-T. Han, V. Ebert, R. Muller, A. Schulz, S. Seeger, J. Wolfrum, J. Arden-Jacob, G. Deltau, N. J. Marx, and K. H. Drexhage, “Design of Multiplex Dyes for the Detection of Different Biomolecules,” 1994 SPIE Proc. 2137:762-774.
K-T. Han, M. Sauer, A. Schulz, S. Seeger, and J. Wolfrum, “Time-Resolved Fluorescence Studies of Labelled Nucleosides,”
Ber. Busenges. Phys. Chem.
97:1728-30 (1993).
K. Chang and R. K. Force, “Time-Resolved Laser-Induced Fluorescence Study on Dyes Used in DNA Sequencing,”
Applied Spectroscopy
47:24-29 (1993).
J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence Lifetime Imaging,”
Analytical Biochemistry
202, 316-330 (1992).
The dyes described in the literature are based on near infrared probes, energy transfer probes to make the intensities equivalent, and other common fluorophores with visible excitation and emission wavelengths. None of these references mentions the use of metal-ligand complexes in determining a base sequence of a nucleotide strand.
The disadvantages of the currently available technology includes nanosecond decay times, which do not allow suppression of prompt auto-fluorescence, limited photostability, small Stoke's shifts and spectral overlap between the absorption and emission spectra.
In addition, with nanosecond decay times it is not possible to reject the auto-fluorescence from the samples, which is especially problematic with the low concentrations involved in the DNA sequencing. Furthermore, the use of nanosecond decay time fluorophores for sequencing based on the decay times, as has been proposed by other laboratories, requires complex instrumentation and is thus not likely to be widely utilized.
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 determining a base sequence of a nucleotide strand. Also, the use of metal-ligand complexes is not mentioned in the previous citations on fluorescence and DNA sequencing.
There remains a need in the art for improved methods of determining a base sequence of a nucleotide strand.
SUMMARY OF THE INVENTION
In accordance with the present invention, a method for determining a base sequence of a nucleotide strand in a sample includes the step of providing a first fragment of the strand. The emission from metal-ligand complexes may be from mixed singlet and triplet states. We will refer to the emission as fluorescence, though a more precise term may be luminescence. A fluorescent metal-ligand complex is coupled to a first oligonucleotide having a sequence complementary to the first fragment to form a first probe. The first probe is added to a sample that contains the first fragment to form a first mixture containing a first reaction product of the first probe and the first fragment. The first mixture is exposed to an exciting amount of radiation, and the fluorescence of the metal-ligand complex is detected. The first base sequence of the first fragment is identified based on fluorescence of the metal-ligand complex. A second fragment of the st
Fredman Jeffrey
Rothwell Figg Ernst & Manbeck
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