Detection of nucleic acid hybridization by fluorescence polarization
First Claim
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1. A method for detecting a nucleic acid, the method comprising:
- contacting a first nucleic acid to a second nucleic acid, which second nucleic acid comprises a neutral or positively charged fluorescent label when hybridized to the first nucleic acid; and
,detecting fluorescence polarization of the resulting mixture of first and second nucleic acids.
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Abstract
Methods, systems and assays are provided for FP detection of nucleic acid hybridization.
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Citations
38 Claims
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1. A method for detecting a nucleic acid, the method comprising:
- contacting a first nucleic acid to a second nucleic acid, which second nucleic acid comprises a neutral or positively charged fluorescent label when hybridized to the first nucleic acid; and
,detecting fluorescence polarization of the resulting mixture of first and second nucleic acids. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
- contacting a first nucleic acid to a second nucleic acid, which second nucleic acid comprises a neutral or positively charged fluorescent label when hybridized to the first nucleic acid; and
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2. A method for detecting a nucleic acid, the method comprising:
contacting a first nucleic acid to a second nucleic acid, which second nucleic acid comprises a neutral or positively charged fluorescent label; and
, detecting fluorescence polarization of the resulting mixture of first and second nucleic acids, wherein the fluorescence polarization is increased by less than about 50% by the addition of polylysine to the first and second nucleic acid.
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25. A method of identifying the presence of a subsequence of nucleotides in a target nucleic acid, the method comprising:
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contacting the target nucleic acid sequence with a labeled nucleic acid probe, which labeled nucleic acid probe comprises a neutral or positively charged label comprising a fluorophore to form a first reaction mixture; and
,detecting the level of fluorescence polarization of the first reaction mixture, wherein the probe comprises the labeled fluorophore when hybridized to the target nucleic acid. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
detecting the level of fluorescence polarization of the plurality of additional reaction mixtures.
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29. The method of claim 28, wherein the plurality of additional target nucleic acids individually comprise at least one locus for a single nucleotide polymorphism.
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30. The method of claim 29, wherein the plurality of additional nucleic acid probes are individually complementary to at least one allele of each of the single nucleotide polymorphisms in the plurality of target nucleic acid sequences.
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31. The method of claim 30, wherein the plurality of additional target nucleic acids are derived from a single species, variety, cultivar, cell, virus, or organism.
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32. The method of claim 31, wherein identification of the single nucleotide polymorphisms provides a single nucleotide polymorphism genotype for the species, variety, cultivar, cell, virus or organism.
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33. The method of claim 25, wherein the fluorescence polarization is increased by less than about 50% by the addition of polylysine to the target and probe nucleic acids.
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34. The method of claim 25, wherein the target and probe nucleic acids are present in a composition which is substantially free of polyion.
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35. The method of claim 34, wherein the composition comprises less than 1 μ
- M polyion.
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36. The method of claim 25, wherein a rotational diffusion rate of a duplex of the target and probe nucleic acids is less than a rotational diffusion rate of the target or probe nucleic acids.
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37. The method of claim 36, wherein the fluorescence polarization of the probe which is duplexed to the target is at least 50% different than the fluorescence polarization of the probe when not duplexed to the target.
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38. The method of claim 25, wherein the target or probe nucleic acids comprise one or more of:
- DNA, RNA, LNA, a DNA analogue, an RNA analogue or a PNA.
Specification