Method and composition for detecting the presence of a nucleic acid sequence in a sample
First Claim
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1. A method for detecting the presence of a nucleic acid sequence in a sample comprising the steps of:
- a) adding a probe labeled with a fluorescent metal-ligand complex to a sample that contains said nucleic acid sequence to form a mixture containing a reaction product;
b) exposing said mixture to an exciting amount of radiation;
c) detecting fluorescence of said metal-ligand complex; and
d) determining presence of said nucleic acid sequence based on fluorescence of said metal-ligand complex, wherein said fluorescent metal ligand complex is selected from the group consisting of [Re(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) (CO)3(isonicotinic acid)]+, [Ru(4,7-diphenyl-1,10-phenanthroline)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline)2(4-methyl,4′
-carboxylic acid-2,2′
-bipyridine)]2+, (Ru(4,7-diphenyl-1,10-phenanthroline (SO3Na)2)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline(SO3Na)2)2(4-methyl,4′
-carboxylic acid-2,2′
-bipyridine)]2+, [Ru(2,2′
-bipyridyl)(1,10-phenanthroline-9-isothiocyanate)], [Os (1,10-phenanthroline)2(5-amino-phenanthroline)]2+, [Os(2,2′
;
6′
,2″
-terpyridine)(Bis(2-diphenylphosphinoethyl)phenyl phosphine)]2+, [Ru bis(2,2′
-bipyridyl)(phenanthroline-maleamide)], [Os(2,2′
-bipyridyl)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)][PF6]2, [Os(2,2′
-bipyridyl)2(4,4′
-N-hydroxysuccidimide ester 2,2′
-bipyridyl)][PF6]2, and [Ru(2,2′
-bipyridyl)2(4,4′
-N-hydroxysuccidimide ester-2,2′
-bipyridyl)]2+.
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Abstract
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. 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. 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.
41 Citations
3 Claims
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1. A method for detecting the presence of a nucleic acid sequence in a sample comprising the steps of:
-
a) adding a probe labeled with a fluorescent metal-ligand complex to a sample that contains said nucleic acid sequence to form a mixture containing a reaction product;
b) exposing said mixture to an exciting amount of radiation;
c) detecting fluorescence of said metal-ligand complex; and
d) determining presence of said nucleic acid sequence based on fluorescence of said metal-ligand complex, wherein said fluorescent metal ligand complex is selected from the group consisting of [Re(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) (CO)3(isonicotinic acid)]+, [Ru(4,7-diphenyl-1,10-phenanthroline)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline)2(4-methyl,4′
-carboxylic acid-2,2′
-bipyridine)]2+, (Ru(4,7-diphenyl-1,10-phenanthroline (SO3Na)2)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline(SO3Na)2)2(4-methyl,4′
-carboxylic acid-2,2′
-bipyridine)]2+, [Ru(2,2′
-bipyridyl)(1,10-phenanthroline-9-isothiocyanate)], [Os (1,10-phenanthroline)2(5-amino-phenanthroline)]2+, [Os(2,2′
;
6′
,2″
-terpyridine)(Bis(2-diphenylphosphinoethyl)phenyl phosphine)]2+, [Ru bis(2,2′
-bipyridyl)(phenanthroline-maleamide)], [Os(2,2′
-bipyridyl)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)][PF6]2, [Os(2,2′
-bipyridyl)2(4,4′
-N-hydroxysuccidimide ester 2,2′
-bipyridyl)][PF6]2, and [Ru(2,2′
-bipyridyl)2(4,4′
-N-hydroxysuccidimide ester-2,2′
-bipyridyl)]2+.
-
-
2. A method for detecting the presence of a nucleic acid sequence in a sample comprising the steps of:
-
a) adding a probe labeled with a fluorescent metal-ligand complex to a sample that contains said nucleic acid sequence to form a mixture containing a reaction product;
b) exposing said mixture to an exciting amount of radiation;
c) detecting fluorescence of said metal-ligand complex; and
d) determining presence of said nucleic acid sequence based on fluorescence of said metal-ligand complex, wherein said metal-ligand complex is capable of emitting polarized fluorescent light after being excited with linearly polarized electromagnetic light energy; wherein said radiation is linearly polarized electromagnetic light energy which causes said mixture to emit polarized fluorescent light;
wherein said detection is measurement of polarization of fluorescent light emission;
wherein prior to forming said reaction product, said oligonucleotide is exposed to said linearly polarized electromagnetic light energy and polarization of fluorescent light is measured; and
wherein said determination of presence of said nucleic acid utilizes the change in polarization of fluorescent light emission of the oligonucleotide and polarization of fluorescent light emission of the mixture.
-
-
3. A composition for detecting the presence of a nucleic acid sequence, comprising a fluorescent metal-ligand complex coupled to an oligonucleotide having a sequence complementary to said nucleic acid sequence, wherein said fluorescent metal ligand complex is selected from the group consisting of [Re(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) (CO)3(isonicotinic acid)]+, [Ru(4,7-diphenyl-1,10-phenanthroline)2(4,4′
- -dicarboxylic acid-2,2′
-bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline)2(4-methyl,4′
-carboxylic acid-2,2′
-bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline (SO3Na)2)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline (SO3Na)2)2(4-methyl,4′
-carboxylic acid-2,2′
-bipyridine)]2+, [Ru(2,2′
-bipyridyl)(1,10-phenanthroline-9-isothiocyanate)], [Os(1,10-phenanthroline)2(5-amino-phenanthroline)]2+, [Os(2,2′
;
6′
,2″
-terpyridine)(Bis(2-diphenylphosphinoethyl)phenyl phosphine)]2−
, [Ru bis(2,2′
-bipyridyl)(phenanthroline-maleamide)], [Os(2,2′
-bipyridyl)2(4,4′
-dicarboxylic acid-2,2′
-bipyridine)][PF6]2, [Os(2,2′
-bipyridyl)2(4,4′
-N-hydroxysuccidimide ester-2,2′
-bipyridyl)][PF6]2, and [Ru(2,2′
-bipyridyl)2(4,4′
-N-hydroxysuccidimide ester-2,2′
-bipyridyl)]2+.
- -dicarboxylic acid-2,2′
Specification
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Current AssigneeJoseph R. Lakowicz
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Original AssigneeJoseph R. Lakowicz
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InventorsLakowicz, Joseph R.
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Primary Examiner(s)Whisenant, Ethan
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Application NumberUS09/007,064Time in Patent Office1,154 DaysField of Search435/3, 536/23.1, 536/24.3, 935/76, 935/77, 935/78US Class Current435/6.11CPC Class CodesC12Q 1/6816 characterised by the detect...C12Q 2561/119 Fluorescence polarisationC12Q 2563/137 Metal/ion, e.g. metal label
