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Kit for detecting nucleic acid targets in samples based on FRET and its application
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A technology for detecting samples and nucleic acid targets, applied in the field of analysis and detection, can solve problems such as increasing detection costs, difficulty in probe design, synthesis, and limiting application scope, and achieves the effects of high sensitivity, rapid homogeneous detection, and good practical value.
Active Publication Date: 2018-09-25
SHENZHEN INST OF ADVANCED TECH
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Problems solved by technology
In order to meet this requirement, Professor Niko Hildebrandt's working group can only synthesize a nucleic acid aptamer composed of DNA and RNA backbones, which greatly increases the cost of detection and the difficulty of probe design and synthesis, and limits the scope of application of this method
At present, there is no research that can solve the problem of circumventing the complex structure of nucleic acid aptamers and switching to kits that only use DNA nucleic acid probes
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Embodiment 1
[0081] This embodiment intends to detect miR-208a in the sample, and its sequence is: 5'-UAGCAGCACGUAAAUAUUGGCG-3';
[0082] The method for detecting miR-208a as above comprises the following steps:
[0083] (1) Preparation of nucleic acid fragments
[0084] (a) Preparation of biotinylated donor nucleic acid fragments:
[0085] The sequence of the biotinylated donor nucleic acid fragment is 5'-TEG-biotin-CGATCAGTCAGGCAA-3';
[0086] (b) Preparation of Cy5-labeled acceptor nucleic acid fragments:
[0087] The sequence of the acceptor nucleic acid fragment is 5'-TTGTGTTCCGATAGGCTAAAAAAA-3', and the 3' end is labeled with Cy5 fluorescent dye;
[0088] (c) preparing the first nucleic acid aptamer combined with the donor nucleic acid fragment:
[0089]The sequence of the nucleic acid ligand is 5'-TTGCCTGACTGATCGCGCCAATATTT-3'.
[0090] (d) prepare the second nucleic acid ligand that binds to the receptor nucleic acid fragment:
[0091] The nucleic acid ligand sequence is 5'-A...
Embodiment 2
[0102] (1) Disperse the biotinylated donor nucleic acid fragment prepared in Example 1, the Cy5-labeled acceptor nucleic acid fragment, the first nucleic acid aptamer, the second nucleic acid ligand, ATP, and SplintR ligase in 1 mL of buffer solution (PBS, pH 7.4), the final concentration is 2nM, and mixed uniformly at room temperature, each nucleic acid fragment has a sequence complementary to each other, and hybridization is formed in the solution. Subsequently, streptavidin (final concentration is 2nM) labeled with L4Tb is added to the solution, because it can specifically bind with the donor nucleic acid labeled with TEG-biotin to obtain the L4Tb-labeled donor nucleic acid fragment, That is, the energy donor in the sensing and detection system.
[0103] (2) Draw the working curve of the biosensor to detect miR-208a
[0104] Add miR-208a at concentrations of 20pM, 100pM, 500pM, 1nM, 1.5nM, and 2nM into the biosensor constructed above, incubate at 37°C for 30 minutes, and m...
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Abstract
The invention discloses an FRET-based (fluorescence resonance energy transfer) kit for detecting nucleic acid target in a sample and application thereof; the kit comprises a donor nucleic acid fragment, a receptor nucleic acid fragment, a first aptamer capable of binding with the receptor nucleic acid fragment, a second aptamer capable of binding with the donor nucleic acid fragment, and SplintR ligase that may bind the first aptamer and the second aptamer. as no need to contain complex DNA-RNA hybrid-structure aptamers, but to be provide, the kit can provide effective accurate detection for microRNA just by being provided with full-DNA single-chain aptamers without containing complex DNA-RNA hybrid-structure aptamers and has better practical value in clinical application, and a method for providing high-sensitivity quick homogenous detection for various biological markers can be provided on such basis.
Description
technical field [0001] The invention belongs to the field of analysis and detection, and relates to a kit for detecting nucleic acid targets in samples based on FRET (fluorescence resonance energy transfer) and an application thereof. Background technique [0002] The rapid development of the national economy has greatly enhanced the public's attention to health. In order to achieve a low cost of health while achieving a high standard of health, there is an urgent need to develop novel diagnostic techniques or drug screening methods. [0003] Fluorescence resonance energy transfer (FRET) refers to the phenomenon of energy transfer between two fluorescent substances at an ultra-short distance (<10nm). The excited donor transfers energy to the acceptor fluorescent substance in a non-radiative way, causing it to emit light. This kind of fluorescence resonance energy transfer has become more and more popular as a simple and fast detection mode in the field of bioanalytical ...
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