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Method for flexibly detecting DNA through exonuclease III assisted multiple circulation amplification

An exonuclease and multiple cycle technology, applied in the field of biological analysis, can solve problems such as limited sensitivity, high background noise of a single cycle signal, and achieve high sensitivity, reduced experimental cost, and good specificity

Inactive Publication Date: 2019-05-03
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its sensitivity is usually limited due to only a single cycle of signal amplification involved and high background noise

Method used

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  • Method for flexibly detecting DNA through exonuclease III assisted multiple circulation amplification
  • Method for flexibly detecting DNA through exonuclease III assisted multiple circulation amplification
  • Method for flexibly detecting DNA through exonuclease III assisted multiple circulation amplification

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preparation example Construction

[0055] In one or some specific embodiments of the present disclosure, the preparation method of the single-stranded DNA binding protein-probe 1 complex and / or the single-stranded DNA binding protein-probe 2 complex comprises the following steps: probe 1 and / or probe 2 and single-stranded DNA binding protein (SSB) in 1 × NEB buffer 4 (the buffer solution is: 50 mmol per liter of potassium acetate, 20 mmol per liter of trimethylol at pH 7.9 aminomethane-acetic acid, 10 mmol / L magnesium acetate and 1 mmol / L dithiothreitol), and incubated at 37°C.

[0056] In one or some specific embodiments of the present disclosure, a method for sensitively detecting DNA by exonuclease III-assisted multiple cycle amplification is provided, and the reaction system of the method includes:

[0057] Exonuclease III (Exo III)-assisted signal amplification is performed in a 20 µl reaction containing target DNA, ssDNA-binding protein-probe 1 complex, and ssDNA-binding protein-probe 2 Complex (a total...

Embodiment 1

[0081] Fluorescence detection: All oligonucleotides were diluted with 10× Tris-ethylenediaminetetraacetic acid (Tris-EDTA) buffer to prepare stock solutions. 1 micromole per liter of hairpin probe (including probe 1 and probe 2 in a molar ratio of 1:1) in annealing buffer (5 mmol per liter of tris-hydrochloric acid at pH 8.0 ( Tris-HCl), 50 mmol per liter of NaCl), incubated at 95°C for 5 minutes, then slowly cooled to room temperature, and stored at 4°C for use. To prepare ssDNA-binding protein (SSB)-probe complexes, two different hairpin probes were mixed with ssDNA-binding protein (SSB) in 1×NEB buffer 4 (50 mmol / L potassium acetate, 20 mmol / L tris-acetic acid pH 7.9, 10 mmol / L magnesium acetate, 1 mmol / L dithiothreitol), incubated at 37°C. Exonuclease III (Exo III)-assisted signal amplification was performed in 20 µl reactions containing different concentrations of DNA, two different ssDNA-binding protein-probe complexes (200 nmol total per liter, the molar ratio is 1:1)...

Embodiment 2

[0083] The principle of the experiment (such as figure 1 ): In this technical scheme, two kinds of hairpin probes (probe 1 and probe 2) with 3′-protruding ends are designed, and the two kinds of probes are modified with the fluorescent group aminofluorescein (FAM) at their 5′-ends. ), the fluorescence of aminofluorescein (FAM) can be efficiently quenched by the consecutive guanine bases opposite it through photoinduced electron transfer (PIET). In the absence of target DNA, since the 3'-protruding end of the probe is resistant to cleavage by Exo III, it cannot be digested by Exo III and detects Very low fluorescence signal. When the target DNA ( figure 1 ), binding to the 3'-overhanging end of probe 1 ( figure 1 ) form a target-probe 1 duplex with a 3'-blunt end that can be recognized and progressively digested by Exonuclease III (Exo III). Thus, the target DNA and trigger primer are released from the duplex. Subsequently, the released target DNA can bind new probe 1 to i...

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Abstract

The invention specifically relates to a method for flexibly detecting DNA through exonuclease III assisted multiple circulation amplification. According to the technical scheme, through combination ofsingle-stranded DNA conjugated protein (SSB) with a barrette probe, the non-specific digestion of exonuclease III (Exo III) is effectively avoided, so that a background signal nearly zero is produced. The existence of a target DNA causes exonuclease III (Exo III) assisted multiple circulation amplification, the sensitivity is high, the detection limit is 3mol / litre, excellent selectivity is achieved, single alkali base mismatch can be recognized, and the method has tremendous potential in the respects of disease diagnosis and biomedical science research.

Description

technical field [0001] The disclosure belongs to the technical field of bioanalysis, and in particular relates to a method for sensitively detecting DNA by exonuclease III-assisted multiple cycle amplification. Background technique [0002] The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art. [0003] Sensitive detection of specific DNA sequences is crucial in the fields of clinical diagnosis, gene therapy, environmental monitoring, and food safety, and various DNA biosensors have been developed. DNA polymerase-based amplification is widely used due to its high sensitivity, and trace amounts of target DNA can be directly amplified to detectable levels by DNA polymerization. Polymerase chain reaction (PCR) is the gold standard for DNA amplification, but it requires expensive thermal cyclers to precisely control the reaction temperature, limiting its widespread use in non-laboratory setting...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
Inventor 张春阳马飞刘文静
Owner SHANDONG NORMAL UNIV
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