High-sensitivity homogeneous DNA hibridization fluorescence detection method
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A fluorescence measurement, high-sensitivity technology, used in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problem of low sensitivity, and achieve the effect of high sensitivity and wide application range
Inactive Publication Date: 2003-02-26
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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But since DTPA-Tb 3+ - The ternary fluorescent complex of salicylic acid is a weak fluorescent complex, and its disadvantage is that the sensitivity is still low, and its lowest detection limit is only 1×10 -9 mol / L
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Embodiment 1
[0032] A DNA probe labeled at the 5'-end (or 3'-end) with a europium complex of EDTA and a DNA probe at the 3'-end (or 5'-end) labeled with a bidentate β-diketone DNA probes are used in DNA hybridization reactions. When the two labeled DNA probes hybridize to the target DNA, EDTA-Eu 3+ Close to bidentate β-diketone to form EDTA-Eu 3+ -β-diketone strongly fluorescent ternary fluorescent complex, the concentration of target DNA can be measured by measuring the fluorescence intensity of the formed ternary fluorescent complex by time-resolved fluorescence assay.
[0033] 1. Synthesis of bidentate β-diketone labels for labeling DNA probes
[0034] This example uses a novel bidentate β-diketone containing a chlorosulfonyl group, 5-(4"-chlorosulfonyl-1',1"-biphenyl-4'-yl)-1,1,1, 2,2-pentafluoro-3,5-pentanedione (CDPP) was used to label DNA probes. The synthesis reaction of CDPP is carried out in two steps, and its reaction principle is shown in the following reaction equation, ...
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Abstract
The present invention provides a high-sensitivity homogeneous DNA hibridization fluorescence determination method. Eu-compound of EDTA is used to label 5'-end or 3'-end DNA probe, a kind of didentate beta-diketone is used to label 5'-end or 3'-end DNA probe for simultaneous hibridization reaction. After two labeled DNA probes are hybridized with target DNA, EDTA-Eu and beta-diketone close to each other to form ternary strong fluorescence compound EDTA-Eu-diketone. By the time identifying fluorescence process, the fluorescence strength of the formed ternary fluorescence compound is measured and the concentration of the target DNa is determined. The present invention has high sensitivty and wide application.
Description
technical field [0001] The invention relates to a homogeneous DNA hybridization assay technology, in particular to a high-sensitivity homogeneous DNA hybridization fluorescence assay by time-resolved fluorescence assay. Background technique [0002] DNA hybridization assay is one of the most widely used methods for infectious disease diagnosis, gene diagnosis, tumor diagnosis, and microbial taxonomy research, as described in the literature, literature 1: S.Inoue, R.Honde, J.Clin.Microbiol., 1990, 28, 1469. Document 2: T. Sekiya, M. Fushimi, H. Hori, S. Hishmura, T. Sugimura, Proc. Natl. Acad. Sci. U.S.A., 1984, 81, 4771. Document 3: I.C. Hsu , R.A. Metcalf, T. Sun, J.A. Welsh, N.J. Wang, C.C. Harris Naure, 1991, 350, 427. However, the traditional DNA hybridization analysis method requires many steps such as stationary phase of the target DNA, pre-hybridization, hybridization, washing, etc., and the operation is complicated and time-consuming. In addition, the hybridization ...
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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 袁景利王桂兰
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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