High-sensitivity homogeneous DNA hibridization fluorescence detection method
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A fluorescence measurement and high-sensitivity technology, applied in the direction of 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: 2005-06-22
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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[0038] 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.
[0039] 1. Synthesis of bidentate β-diketone labels for labeling DNA probes
[0040] 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 invention provides a high-sensitivity homogeneous DNA hybridization fluorescence assay method. It uses a europium complex of ethylenediaminetetraacetic acid to label a 5'- or 3'-terminal DNA probe, and a bidentate β-diketone to label a 3'- or 5'-terminal DNA probe, At the same time, it is used for DNA hybridization reaction; when the two labeled DNA probes hybridize with the target DNA, EDTA-Eu and β-diketone approach each other to form a strong fluorescent ternary fluorescent complex of EDTA-Eu-β-diketone, The concentration of the target DNA can be measured by measuring the fluorescence intensity of the formed ternary fluorescent complex by time-resolved fluorescence method. The method of the invention has higher sensitivity and wide application range.
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 Patents(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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