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A method and kit for detecting the 3'-5' exo-cutting activity of nucleases on specific bases

A nuclease and kit technology, which is used in the field of detecting the 3'-5' exocytosis activity of nucleases on specific bases, can solve the problem that radioisotope methods are prone to pollution, difficult to achieve high-throughput, automated, and inaccurate detection. Problems such as exonuclease activity

Active Publication Date: 2022-06-07
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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  • A method and kit for detecting the 3'-5' exo-cutting activity of nucleases on specific bases
  • A method and kit for detecting the 3'-5' exo-cutting activity of nucleases on specific bases
  • A method and kit for detecting the 3'-5' exo-cutting activity of nucleases on specific bases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Establishment of a method for detecting the 3'-5' exonucleation activity of nucleases on specific bases

[0049] 1. Detection principle

[0050] like figure 1 shown,

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

[0052] Design a primer that is partially complementary to the known sequence according to the experimental requirements. In the primer, the phosphodiester bond between the penultimate base and the penultimate base in the 5'-3' sequence is modified by sulfurylation , the 5' end is modified by phosphorylation, and the la...

Embodiment 2

[0080] Embodiment 2, detect nuclease to specific base 3 '-5 ' exonucleation activity

[0081] In this example, exonuclease I was used as the internal reference nuclease with known activity (the activity size was 10 U / ul), and the phi29 DNA polymerase of Enzymatic and the phi29 DNA polymerase of BGI were detected according to the second method of Example 1 when there was no polymerization reaction. Desired 3'-5' exo-active size in the case of dNTPs for unmodified C bases in the case of correct pairing. During the test, two control groups were set at the same time to prevent the tested enzyme from cutting out the bases completely or cutting all the bases on the chip cleanly, resulting in inaccurate results.

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

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

[0084] Table 1

[0085]

[0086] Specific steps are as follows:

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

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Abstract

The invention discloses a method and a kit for detecting the 3'-5' excision activity of a nuclease on a specific base. The present invention proposes for the first time to detect the exolytic activity of an enzyme to a specific base; any base can be detected by using the invention, including dATP, dCTP, dGTP, dTTP, dUTP and degenerate bases, etc., and the application range is very wide; the invention can Detects the 3'-5' exoactivity of most nucleases; uses fluorescence as the detection signal, avoids radioactive contamination, and has high sensitivity; does not require fluorophore-modified nucleotide chains, and has low cost; can be used for rapid and large-scale Screen nucleases with flexible applications and high throughput.

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' exocytosis activity of a nuclease on a specific base. Background technique [0002] Many nucleases have 3'-5' exonucleation activity. Detecting the 3'-5' exonuclease activity of nucleases is of great significance for understanding the proofreading 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 in the case of correct pairing and different forms of wrong pairing. Studying the exonucleation activity of nucleases on a specific base is of great significance to molecular biology research and drug development. [0003] Existing techniques for detecting 3'-5' exonuclease activity mostly require radiolabeling, gel electrophoresis, enzy...

Claims

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Application Information

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Patent Type & Authority Patents(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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