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Method for specific regulation and control on activity of nuclease

A technology of nuclease activity and specificity, applied in the field of DNA sequence detection, can solve the problems of interfering with the normal operation of the enzyme-nucleic acid probe system, unclear nuclease distribution mechanism, and reduced detection efficiency, so as to achieve stable detection efficiency and retention The main activity and the effect of improving detection efficiency

Active Publication Date: 2020-05-19
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Through the above analysis, the existing problems and defects of the prior art are: (1) The phenomenon of single-strand cutting by nucleases is ubiquitous and seriously interferes with the normal work of the enzyme-nucleic acid probe system, reducing the detection efficiency;
[0005] (2) This phenomenon is difficult to predict, and the current method can only blindly optimize the probe sequence to avoid self-cleavage of single-stranded probes by nucleases as much as possible, resulting in extremely high costs
[0006] The difficulty in solving the above problems and defects is: the distribution mechanism of nucleases on DNA double strands and DNA single strands is still unclear

Method used

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  • Method for specific regulation and control on activity of nuclease
  • Method for specific regulation and control on activity of nuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Check the inhibitory effect of double-stranded DNA on nuclease side activity

[0065] In this example, a kit containing only Endo IV / APE1 / lambda exo, single-stranded probe, buffer, and H 2 Add double-stranded DNA irrelevant to the reaction system to the system of O to test whether double-stranded DNA can inhibit nuclease from cutting single-stranded probes. The specific implementation steps are as follows:

[0066] 1. Design the reaction system and synthesize the oligonucleotide chain.

[0067] The reaction system includes nuclease, probe, buffer, H 2 O and double-stranded DNA. The sequences of primers and probes are listed in Table 1.

[0068] Table 1, check the inhibitory effect of double-stranded DNA on nuclease side activity

[0069]

[0070] 2. Configure the reaction system

[0071] Add 100nM probe, 5uL ThermoPol buffer (10×), 0.1 unit EndoIV or 2 units APE-1 or 2 units lambda exo to 400uL enzyme labeling strip, and make up to 50uL total volu...

Embodiment 2

[0076] Embodiment 2: Guiding chain improves the existing detection technology.

[0077] In this example, the Guiding chain was added to the nucleic acid detection system participated by Endo IV, and the improvement of the detection performance by the Guiding chain was tested. For detection principle see figure 1 , the specific implementation steps are as follows:

[0078] 1. Design the reaction system and synthesize the oligonucleotide chain.

[0079] The target is the EGFR-L858R mutation. Based on the DNA sequence of the mutation, design the substrate chain (MT, WT), probe, blocker and corresponding Guiding required for the nucleic acid detection system involved in Endo IV chain. The sequences of primers and probes are listed in Table 2. The schematic diagram of the probe system is shown in figure 1 .

[0080] Table 2, Guiding Chain Improvements to Existing Detection Techniques

[0081]

[0082] 2. Configure the reaction system

[0083] In 400uL enzyme labeling s...

Embodiment 3

[0088] Example 3: Detection of low-abundance mutations

[0089] In this embodiment, the system to be tested is a mixed system of wild chain and mutant chain, and a series of different mutation ratios are set to detect the lowest possible mutation ratio. For detection principle see figure 1 , the specific implementation steps are as follows:

[0090] 1. Design the reaction system and synthesize the oligonucleotide chain.

[0091] The target is the EGFR-L858R mutation. According to the DNA sequence of the mutation, design the substrate strands (MT, WT), probes, blockers and corresponding Guiding required for the nucleic acid detection system involved in Endo IV chain. The sequences of primers and probes are listed in Table 2. The schematic diagram of the probe system is shown in figure 1 .

[0092] 2. Configure the reaction system

[0093] 50μL reaction system of mutants with different proportions: probe amount: 1000nM; mutation ratio: 100%, 10%, 1%, 0.5%, 0.1%, 0.01%, ...

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Abstract

The invention belongs to the technical field of DNA (deoxyribonucleic acid) sequence detection and discloses a method for specific regulation and control on activity of nuclease. The method comprisesthe following steps: designing a corresponding Guiding chain for gene sequences at different mutant loci; synthesizing and preparing an expected Guiding chain; mixing the Guiding chain with a probe detection system, and setting the ratio of the Guiding chain to a substrate chain to 2:1; and adding Endo IV into the mixed system, converting the substrate chain into a longer DNA double-chain, and performing real-time fluorescence testing. By adopting the method, the activities of an incision enzyme IV / APE-1 / lambda and an excision enzyme are regulated and controlled, background signals generated since the secondary activity of the nuclease can be reduced, meanwhile, the main activity that a target sequence is cut by the nuclease is maintained to the maximum extent, and thus the detection efficiency can be improved.

Description

technical field [0001] The invention belongs to the technical field of DNA sequence detection, and in particular relates to a method for specifically regulating nuclease activity. Background technique [0002] Nucleases are currently widely used tools in biological, chemical and medical research. They are widely used in the development of biosensors, gene editing technology and new drugs. Different nucleases have different cleavage properties and corresponding substrates are also different. At the level of practical application, the current application of nuclease is based on its predictable working mechanism. For example, we usually want Endo IV to be able to stably and specifically cut DNA duplexes with gaps. However, nucleases are easily interfered by other nucleic acid strands in the system, and their cleavage properties are not stable. Especially for nucleases that target double-stranded DNA, they often cleave single-stranded nucleic acids non-specifically. This ph...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/682C12Q1/6827
CPCC12Q1/6858C12Q1/682C12Q1/6827C12Q2521/301C12Q2521/319C12Q2525/161C12Q2525/186C12Q2527/127C12Q2563/107
Inventor 肖先金明志浩章伟
Owner HUAZHONG UNIV OF SCI & TECH
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