Nucleic acid analysis method based on constant-temperature cross-catalyzed nuclease reaction

A nucleic acid analysis and nuclease technology, applied in the field of nucleic acid analysis based on constant temperature cross-catalyzed nuclease reaction

Active Publication Date: 2020-04-07
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Protease-mediated cross-catalyzed DNA amplification systems have been developed for in vitro bioanalysis, however, enzyme-free cross-catalyzed amplification machines that can be used for in vivo analysis, especially those involving HCR, have been rarely investigated

Method used

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  • Nucleic acid analysis method based on constant-temperature cross-catalyzed nuclease reaction
  • Nucleic acid analysis method based on constant-temperature cross-catalyzed nuclease reaction
  • Nucleic acid analysis method based on constant-temperature cross-catalyzed nuclease reaction

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Experimental program
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Effect test

Embodiment 1

[0034] Design of DNA probes: Use NUPACK software to design relevant DNA 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. However, when the target exists, it can trigger a constant temperature cross-catalyzed nuclease reaction and generate fluorescence. Resonance energy transfer to realize the detection of target objects. All DNA 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 DNA probes to 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.

[0035] Fig. 1 (1) is a schematic diagram of a thermostatic cross-catalyzed nuclease reaction for ...

Embodiment 2

[0039] DNA detection based on isothermal cross-catalyzed nuclease reaction

[0040] In hydroxyethylpiperazine ethyl sulfonate buffer (concentration is 10mM, pH 7.2, containing 1M NaCl and 50mM MgCl 2 ), all DNA reactions (H 1 、H 2 、H 3 、H 4 , S and L are 200nM) and different concentrations of priming chain T (0, 1 × 10 -11 , 5×10 -11 , 1×10 -10 , 5×10 -10 , 1×10 -9 , 5×10 -9 , 1×10 -8 , 2.5×10 -8 , 5×10 -8 M) mixing, incubating at room temperature for 3 h, and measuring the fluorescence intensity of the system using a fluorescence spectrometer (excitation voltage 600V, excitation slit 5nm, emission slit 10nm, excitation wavelength 490nm, wavelength scanning range 505-650nm).

[0041] Depend on image 3 (A) It can be seen that when no target DNA is added to the R-HCR system, each DNA probe can maintain its own stability, and the fluorescence of the system only changes slightly ( image 3 Curve a) in (A), when adding different concentrations of target DNA, the chan...

Embodiment 3

[0045] In vitro detection of miRNA-21 based on isothermal cross-catalyzed nuclease reaction

[0046] In hydroxyethylpiperazine ethyl sulfonate buffer (concentration is 10mM, pH 7.2, containing 1M NaCl and 50mM MgCl 2 ), all DNA reactions (H 1 、H 2 、H 3 、H 4 , S, L are all 200nM, H 5 100nM) 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 , 3×10 -8 , 5×10 -8 M) mixing, incubating at room temperature for 3 h, and measuring the fluorescence intensity of the system using a fluorescence spectrometer (excitation voltage 600V, excitation slit 5nm, emission slit 10nm, excitation wavelength 490nm, wavelength scanning range 505-650nm).

[0047] Figure 1 (4) is a schematic diagram of the constant temperature cross-catalyzed nuclease reaction for miRNA-21 detection. Depend on Figure 4 (A) It can be seen that when miRNA-21 is not added to the R-HCR system, each DNA probe can maintain its own stability, and...

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Abstract

The invention discloses a nucleic acid analysis method based on a constant-temperature cross-catalyzed nuclease reaction. A metastable hairpin in an HCR amplifier is activated by a target to be openedautonomously and continuously; a double-stranded DNA nanowire containing a large amount of DNAzyme is generated; and the DNAzyme continuously catalyzes a shearing substrate to release a large amountof new initiation chains to reversely activate an HCR reaction. Due to the facts that the DNAzyme can continuously accumulate HCR initiation chains and HCR can continuously assemble a large amount ofDNAzyme, the cross-catalytic amplification reaction can convert the molecular recognition process into significantly amplified signals to be output. The R-HCR amplifier can be easily combined with anauxiliary sensing module to be developed into a universal sensing system, and ultra-sensitive in-vitro detection and intracellular imaging of single-molecule miRNA are successfully achieved. The nucleic acid analysis method provided by the invention has wide application prospect in the aspect of in-vivo detection of trace biomarkers in cytobiology and clinical diagnosis.

Description

technical field [0001] The invention belongs to the field of molecular detection, in particular to a nucleic acid analysis method based on constant temperature cross-catalyzed nuclease reaction. Background technique [0002] Nucleic acid constant temperature amplification system has become a research hotspot because of its wide application in clinical diagnosis, food safety, environmental monitoring and forensic analysis. Based on different catalytic reaction mechanisms, these strategies are mainly classified into enzymatic amplification and enzyme-free amplification methods. These enzymatic machinery include rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and strand displacement amplification (SDA). These enzymatic machinery often rely on certain proteases for their effectiveness, which are easily disturbed by the external environment. Therefore, there is an urgent need to develop more powerful and convenient sensing strategies for the an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/6841C12Q1/6886
CPCC12Q1/682C12Q1/6841C12Q1/6886C12Q2600/158C12Q2600/178C12Q2521/345C12Q2525/207C12Q2525/301C12Q2563/107
Inventor 王富安魏洁李丰哲
Owner WUHAN UNIV
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