Method for detecting 3'-5' exo-activity of specific base by nuclease and kit thereof

A nuclease and kit technology, which is applied in the field of detecting the 3'-5' exocytosis activity of nucleases on specific bases, can solve the problems of limiting the scope of modified bases, complicated operation, long cycle, etc., to avoid radioactive contamination, Wide range of applications and flexible effects

Active Publication Date: 2019-04-19
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The radioisotope method is prone to pollution, and the operation is complicated and the cycle is long, so it is difficult to achieve high-throughput and automation
In the patent CN104293930 A, the detection results of enzymes with particularly high or low exolytic activity cannot be very accurate, so it has certain limitations, and at the same time, it cannot accurately detect the exolytic activity of nucleases on a specific base
Patent CN104293917 B needs to synthesize a probe with a stem-loop structure and a quencher group and a fluorescent group, which is costly and also unable to detect the excision activity of a nuclease on a specific base, especially because the 3' end One base bears a fluorophore, limiting the range of modified bases that can be detected

Method used

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  • Method for detecting 3'-5' exo-activity of specific base by nuclease and kit thereof
  • Method for detecting 3'-5' exo-activity of specific base by nuclease and kit thereof
  • Method for detecting 3'-5' exo-activity of specific base by nuclease and kit thereof

Examples

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Embodiment 1

[0048] Example 1. Establishment of a method for detecting the size of nuclease exonucleating activity to a specific base 3'-5'

[0049] 1. Detection principle

[0050] Such as figure 1 as shown,

[0051]1) Use a single-stranded DNA molecule immobilized on a solid phase with a known sequence as a template, such as oligonucleotides immobilized on magnetic beads with streptavidin and biotin or spotted on Oligonucleotides on the chip, etc., the exposed end of the single-stranded DNA molecule containing a known sequence needs to be modified to prevent exonuclease digestion, such as phosphorylation modification, sulfur modification, etc. (such as figure 1 a);

[0052] According to the experimental requirements, design a primer partially complementary to the known sequence, in which the phosphodiester bond between the penultimate base and the penultimate base in the order of 5'-3' is modified by sulfurylation , the 5' end is modified by phosphorylation, and the last base at the 3...

Embodiment 2

[0080] Example 2, detection of nuclease exonuclease activity on specific base 3'-5'

[0081] This embodiment uses exonuclease I as the internal reference nuclease of known activity (activity size is 10U / ul), detects the phi29DNA polymerase of Enzymatic and the phi29DNA polymerase of BGI according to the second method of embodiment 1 when there is no polymerization reaction In the case of the desired dNTP, the size of the 3'-5' exolytic activity of the unmodified C base under the condition of correct pairing. During the testing process, two control groups are set at the same time to prevent inaccurate results caused by the detected enzyme not cutting out the bases at all or cutting out all the bases on the chip.

[0082] Equipment used in the following method: BGISEQ-500 sequencer

[0083] The reagents used in the following method are shown in Table 1 below:

[0084] Table 1

[0085]

[0086] Specific steps are as follows:

[0087] 1. Synthesize primers partially complem...

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Abstract

The invention discloses a method for detecting 3'-5' exo-activity of a specific base by nuclease and a kit thereof. The method firstly detects the exo-activity of the specific base by enzyme; the method can detect any base, including dATP, dCTP, dGTP, dTTP, dUTP and degenerate base, and the like, and the application range is wide; the method can detect the 3'-5' exo-activity of most nucleases; theuse of fluorescence as a detection signal avoids radioactive contamination and has high sensitivity; nucleotide chains modified by fluorophores cannot be required, and the cost is low; the method canbe used to rapidly and extensively screen nucleases, and the application is flexible and the flux is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a kit for detecting the 3'-5' excision activity of a nuclease on a specific base. Background technique [0002] Many nucleases have 3'-5' exo-cutting activity. Detection of nuclease 3'-5' exonuclease activity is of great significance for understanding the correction ability of DNA polymerase and the degradation rate of DNA exonuclease. Nucleases have different exo-cutting abilities to different bases (including various modified bases, degenerate bases, etc.), and also have different exo-cutting abilities to bases under correct pairing and different forms of wrong pairing. Studying the exoclease activity of nucleases on a specific base is of great significance to molecular biology research and drug development. [0003] Most of the existing techniques for detecting 3'-5' exonuclease activity require radioactive labeling, gel electrophoresis, enzyme-linked im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816C12Q1/34
CPCC12Q1/34C12Q1/6816G01N2333/922C12Q2563/107C12Q2521/101C12Q2525/186
Inventor 李长英章文蔚陈奥徐崇钧龚梅花王静静罗银玲罗宇芬
Owner MGI TECH CO LTD
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