Nucleotide composition, kit and detection method

A composition and nucleotide technology, applied in the field of molecular biology, can solve the problems of complex operation of gene chip technology, and achieve the effects of saving experimental costs, increasing specificity, and reducing false positives

Active Publication Date: 2019-02-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex operation of gene chip technology, after the PCR reaction process, steps such as cleaning, sealing, and color development are still required to distinguish the results. The cumbersome steps limit the routine application of these methods in the laboratory.

Method used

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  • Nucleotide composition, kit and detection method
  • Nucleotide composition, kit and detection method
  • Nucleotide composition, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the establishment of DNA silver nanocluster fluorescent visual detection technology

[0047] The process of constructing the DNA silver nanocluster fluorescent visual detection technology in this embodiment includes composition design, formation of silver nanocluster probes, asymmetric PCR process, hybridization of PCR products and silver nanocluster probes.

[0048] (1) Design of composition for fluorescent visual detection of DNA silver nanoclusters

[0049] The present invention provides a nucleotide composition, including composition 1 and composition 2. Composition 1 includes a sequence consisting of a silver nanocluster nucleation sequence and a recognition sequence, which is used to combine with silver nanoions to form silver nanoclusters ; Composition 2 includes a sequence consisting of a hybridized complementary sequence and a recognition sequence of the silver nanocluster excitation sequence, the recognition sequence in the composition 1 and the ...

Embodiment 2

[0076] Example 2. Establishment of DNA silver nanocluster fluorescent visual detection technology for the 35S promoter of transgenic maize MON810

[0077] (1) Composition design

[0078] In this example, according to the partial nucleotide sequence of the exogenous gene screening element 35S promoter of transgenic maize MON810, a composition for visual detection of DNA silver nanocluster fluorescence as shown in Table 2 was designed.

[0079] Table 2

[0080]

[0081] AG-F in Table 2 includes the hybridization complementary sequence, recognition sequence (italicized part) and primer 1 of the silver nanocluster excitation sequence in the composition 2 of Example 1 in order from the 5' end; AG-R is primer 2 Specifically, the complementary hybridization sequence of the silver nanocluster excitation sequence is 5'-CCCCACCCCACCCCACCC-3' (the nucleotide sequence shown in SEQ ID No. 4 in the sequence table), and the recognition sequence is 5'-TATAATAAATTTTAAATATTATTTATTAAT-3' (se...

Embodiment 3

[0090] Example 3. Optimization of technical conditions for the fluorescent visual detection of DNA silver nanoclusters for the 35S promoter of transgenic maize MON810

[0091] (1) Optimization of incubation time in the dark during the preparation of silver nanocluster probes

[0092] The experiment was carried out in 3 groups, and the steps for preparing silver nanocluster probes were the same as those described in Example 1 (two), except that the incubation at room temperature and in the dark as described in step ⑥ were successively set to three groups of 14h, 10h, and 8h; Wherein the composition one is specifically the composition one shown in Table 2.

[0093] Directly replace the reaction system after the completion of the asymmetric PCR reaction in the process described in Example 1 (four) with the artificially synthesized single-stranded product containing the excitation sequence, and hybridize with the silver nanocluster probes prepared by the above-mentioned 3 groups o...

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Abstract

The invention discloses a nucleotide composition, a kit containing nucleotide and a detection method. When a sample to be detected contains target molecules to be detected, a large amount of single-chain products can be obtained through subjecting a composition II and a primer pair, which are designed in the invention, to an asymmetric PCR, these single-chain products can be hybridized with a silver nanocluster probe prepared by using a composition I designed in the invention and then can emit fluorescence, and thus, the visible detection on the target molecules to be detected is achieve; thedisadvantage that the traditional methods are dependent on instrument discrimination is overcome, a visualized qualitative quantitative novel detection method which is independent of complicated instruments is established, the existing detection techniques and methods are enriched, and thus, a way of think and a technical base are provided for applying silver nano clusters in the aspect of transgenosis in the future. In a specific embodiment, the compositions and the method, disclosed by the invention, have the sensitivity of 0.5ng per microliter and are good in repeatability and high in specificity.

Description

technical field [0001] The present application relates to the technical field of molecular biology, in particular, to a nucleotide composition, a kit containing the nucleotide composition and a detection method. Background technique [0002] In July 2002, my country began to implement the "Regulations on the Sanitation Management of Genetically Modified Foods" to manage the labeling of genetically modified food products. At present, the detection methods of genetically modified products commonly used in the world mainly include enzyme-linked immunoassay (ELISA), PCR-electrophoresis detection, fluorescent quantitative PCR detection and other technologies. High, and the data needs to be analyzed before the results can be obtained. Therefore, the visibility detection technology for genetically modified crops has entered people's field of vision. Deng Tingting and others have used the visual chip method to detect Mir162 in genetically modified corn, and the detection limit can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895
Inventor 许文涛罗云波牛晨启黄昆仑徐瑗聪杜再慧贺晓云
Owner CHINA AGRI UNIV
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