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A Nucleic Acid Analysis Method Based on Cascade Hybridization Chain Reaction

A hybrid chain reaction, cascade technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as unresearched

Active Publication Date: 2020-03-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Two-level HCR has further amplification on the basis of single-level HCR, which has not been studied

Method used

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  • A Nucleic Acid Analysis Method Based on Cascade Hybridization Chain Reaction
  • A Nucleic Acid Analysis Method Based on Cascade Hybridization Chain Reaction
  • A Nucleic Acid Analysis Method Based on Cascade Hybridization Chain Reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Design of hairpin probes: Use NUPACK software to design relevant hairpin probes, and entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize relevant nucleic acid sequences. When there is no target, ensure that the stem end of each hairpin has enough complementary base pairing to maintain its own stability and keep the fluorescence unchanged, but when there is a target, it can trigger a cascade hybridization chain reaction and form a branch. Shaped DNA nanostructures undergo fluorescence resonance energy transfer to achieve target detection. All hairpin probe dry powders were first dissolved in phosphate buffer, and their absorbance was measured with a UV spectrophotometer to calculate the exact concentration. 2 ) Prepare all the hairpin probes at 4 μM, in PCR at 95° C. for 5 minutes, and at 25° C. for 2 hours to form stable hairpins. All reactions were carried out in HEPES buffer.

[0043] figure 1 A is the schematic diagram of cascade hybridization chain r...

Embodiment 2

[0046] [Example 2] DNA detection based on cascade hybridization chain reaction

[0047] In hydroxyethylpiperazine ethyl sulfonate buffer (concentration is 10mM, pH 7.2, containing 1M NaCl and 50mM MgCl 2 ), set all hairpin probes (H 1 、H 2 、H 3 、H 4 、H 5 and H 6 Both are 200nM) with different concentrations of target DNA (0, 1×10 -11 , 5×10 -11 , 1×10 -10 , 5×10 -10 , 1×10 -9 , 5×10 -9 , 1×10 -8 , 5×10 -8 , 1×10 -7 M) Mixing, incubating at room temperature for 2h, using a fluorescence spectrometer to measure the fluorescence intensity of the system (excitation voltage 600V, excitation slit 5nm, emission slit 10nm, excitation wavelength 490nm, wavelength scanning range 505-650nm).

[0048] Depend on figure 2 (B) It can be seen that when no target DNA is added to the C-HCR system, each hairpin probe can maintain its own stability, and the fluorescence of the system will not change ( figure 2 Curve a) in (B), when different concentrations of target DNA are added...

Embodiment 3

[0051] [Example 3] In vitro detection of miRNA-21 based on cascade hybridization chain reaction

[0052] In hydroxyethylpiperazine ethyl sulfonate buffer (concentration is 10mM, pH 7.2, containing 1M NaCl and 50mM MgCl 2 ), set all hairpin probes (H 1 、H 2 、H 3 、H 4 、H 5 、H 6 Both are 200nM, H 7 50nM) with different concentrations of miRNA-21 (0, 1×10 -11 , 5×10 -11 , 1×10 -10 , 5×10 -10 , 1×10 -9 , 5×10 -9 , 1×10 -8 , 5×10 -8 , 1×10 -7 M) Mixing, incubating at room temperature for 2h, using a fluorescence spectrometer to measure the fluorescence intensity of the system (excitation voltage 600V, excitation slit 5nm, emission slit 10nm, excitation wavelength 490nm, wavelength scanning range 505-650nm).

[0053] figure 1 B is the schematic diagram of cascade hybridization chain reaction for miRNA-21 detection. Depend on Figure 4 (A) It can be seen that when miRNA-21 is not added to the C-HCR system, each hairpin probe can maintain its own stability, and the fl...

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Abstract

The invention discloses a nucleic acid analyzing method based on cascade HCR (hybridization chain reaction), and belongs to the field of molecule detection. The nucleic acid analyzing method is characterized in that on the basis of single HCR, two stages of HCR reaction are designed, and the signals can be further amplified on the basis of single HCR; the hairpin nucleic acid probes marked with multiple different fluorescent dyes can be alternatively hybridized by a final target product, so as to form a branched DNA (deoxyribonucleic acid) nanometer structure; the signal output is provided bythe fluorescent resonance energy transfer, and the concentration of the target nucleic acid can be judged according to the change value of the fluorescence intensity of fluorophore. The nucleic acid analyzing method has the advantages that the nucleic acid analyzing method can be used for in-vitro detection of DNA and miRNA (miniature ribonucleic acid), the specificity is good, and the sensitivityis high; the nucleic acid analyzing method can be used for the imaging analysis of miRNA in cells, and be favorable for the early diagnosis on cancers.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a nucleic acid analysis method based on cascade hybridization chain reaction. Background technique [0002] Small molecular nucleic acid (miRNA) is a kind of tumor marker closely related to cancer. It is composed of small molecule non-coding RNA of 18-25 nucleotides. Play an important role in the process (Lee Y.S., Dutta A., Annu. Rev. Pathol., 2009, 4, 199-227.). miRNA is tissue-specific and abnormally expressed in the serum of cancer patients. Its expression is closely related to cancer classification, type and stage, and has high sensitivity, stability and specificity. Therefore, the establishment of a simple and sensitive miRNA detection method is of great significance for the early diagnosis of major diseases and the development of related drugs. [0003] At present, the detection methods of miRNA mainly include Northern blot, microarray hybridization, non-is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6818C12Q1/6886
Inventor 王富安魏洁
Owner WUHAN UNIV
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