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A method for specific regulation of nuclease activity

A technology with nuclease activity and specificity, applied in the field of DNA sequence detection, can solve the problems of normal operation of the interferase-nucleic acid probe system, unclear distribution mechanism of nuclease, and reduced detection efficiency, so that the detection efficiency can be kept stable and preserved. The main activity and the effect of improving the detection efficiency

Active Publication Date: 2022-03-08
HUAZHONG UNIV OF SCI & TECH
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  • 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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  • A method for specific regulation of nuclease activity
  • A method for specific regulation of nuclease activity
  • A method for specific regulation of nuclease activity

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. Double-stranded DNA and probe sequences 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 a total vol...

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. 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 .

[0092] 2. Configure the reaction system

[0093] 50 μL reaction system of mutants with different proportions: probe amount: 1000nM; mutation ratio: 100%, 10%, 1%, 0.5%, 0.1%, 0.01%, 0%. The...

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Abstract

The invention belongs to the technical field of DNA sequence detection, and discloses a method for specifically regulating nuclease activity, designing corresponding Guiding chains for gene sequences at different mutation sites; synthesizing and preparing the required Guiding chains; Mix with the probe detection system so that the ratio of Guiding strand to substrate strand is 2:1; add Endo IV to the above mixed system to convert the substrate strand into a longer DNA double strand, and perform real-time fluorescence measurement. The regulation of endonuclease IV / APE-1 / lambda exonuclease activity involved in the present invention reduces the background signal due to nuclease side activity, and at the same time retains the main activity of nuclease cutting the target sequence to the greatest extent, thereby improving detection efficacy.

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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Patent Type & Authority Patents(China)
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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