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T7EI-like nuclease reaction buffer system, DNA mismatch detection method and application thereof

A buffer system and nuclease technology, applied in biochemical equipment and methods, microbial assay/inspection, etc., can solve problems such as affecting application, easy omission of SNP type differences, biased results, etc. Effects of the ability to mismatch DNA

Pending Publication Date: 2020-10-09
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] T7EI is a multifunctional nuclease. Although the enzyme is widely used in mutation screening detection, some characteristics of the enzyme affect the application of the enzyme.
First of all, there are obvious differences in the recognition efficiency of the enzyme for different mismatched substrates. It has the highest recognition and cleavage efficiency for Holiday DNA, followed by InDel-type mismatches. Its recognition effect for SNP-type mismatches is poor, and for mismatched bases The category has a certain preference [3]
In practical applications, InDel and SNP type mismatches exist at the same time, making it easy to miss SNP type differences in the detection results. Even if some SNP type differences are detected, the results are biased

Method used

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  • T7EI-like nuclease reaction buffer system, DNA mismatch detection method and application thereof
  • T7EI-like nuclease reaction buffer system, DNA mismatch detection method and application thereof
  • T7EI-like nuclease reaction buffer system, DNA mismatch detection method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] This embodiment provides a reaction buffer system for T7EI-like nuclease: including T7EI-like nuclease, Tris-HCl and Tris-Ac that provide buffering capacity, detergent Triton X-100 and Tween that enhance the dispersion of enzyme protein -20, metal ion Mg involved in substrate binding and catalysis 2+ 、Co 2+ , Mn 2+ etc., including non-specific proteins such as BSA that enhance enzyme stability, including DNA ligase that counteracts non-specific cleavage activity, etc.

[0030] The above-mentioned T7EI-like nucleases include T7EI (T7 endonuclease I) and P-SSP7EI (P-SSP7endonuclease I) and their homologous proteins; the above-mentioned proteins can be from commercial products, can be obtained by expression and purification, can be the enzyme protein itself, or It can be a fusion protein of T7EI-like protein, such as T7EI fused with maltose binding protein (MBP), and its activity needs to be calibrated before application;

[0031] The buffer capacity provider is Tris-HC...

Embodiment 2

[0038] This embodiment provides a method for DNA mismatch recognition, using the reaction buffer system of Example 1 to enhance the cleavage ability of T7EI-like nuclease, including the following steps:

[0039] After the reaction buffer is prepared according to the method of Example 1, the sample is pretreated firstly: the PCR product or the PCR product mixture is denatured and annealed. The denaturing annealing process is shown in Table 1. Then carry out precipitation redissolution, add 1 / 10 volume of 3M potassium acetate solution (pH 5.2), add 2 times the volume of pre-cooled absolute ethanol, place on ice for 30min, centrifuge at 13000g 4°C for 10min, carefully discard the supernatant, add 1ml 70 The precipitate was washed with % ethanol, centrifuged at 13,000 g at 4°C for 10 min, the supernatant was carefully discarded, dried at room temperature, and TE buffer was added to dissolve the precipitate to obtain a mismatched substrate. If the detected mismatch type is InDel t...

Embodiment 3

[0046] This embodiment discloses the application of an enhanced reaction buffer system in various practical scenarios.

[0047] scene 1:

[0048] In the analysis of gene editing efficiency, first perform PCR amplification, denaturation annealing, precipitation and redissolution, then perform enzyme digestion according to the operation in Example 2, and then use gel electrophoresis to separate, and use gel imaging software to analyze the electrophoretic bands. The brightness is analyzed, and the efficiency of gene editing is calculated based on it. Calculated as follows:

[0049] Editing efficiency (%)=100x[1-(1-cuttable DNA ratio) 1 / 2 ]

[0050] Since the T7EI-like nuclease in the reaction buffer system disclosed by the present invention can better recognize SNP type mismatches, the proportion of cleavable DNA obtained is more accurate, and the gene editing effect evaluation based on it is also more accurate.

[0051] Scenario 2:

[0052] Screening of gene-edited individu...

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Abstract

The invention discloses a T7EI-like nuclease reaction buffer system, a DNA mismatch detection method and an application thereof, and relates to a reaction buffer solution formula of T7EI-like nucleaseand an application of the buffer system in enhancing mismatch recognition cutting capacity of the nuclease. In a conventional reaction buffer system, T7EI-like nuclease has a relatively good recognition and cleavage capability on mismatched DNA caused by insertion and deletion (InDel), and has a relatively poor recognition and cleavage capability on mismatched DNA caused by single nucleotide polymorphism (SNP); in the reaction buffer system provided by the invention, the T7EI-like nuclease has good recognition and cleavage capability on two types of mismatched DNAs. The core of the reaction buffer system is the specific buffer solute ratio, detergent concentration and divalent metal ion and auxiliary protein ratio. The method can be applied to detection of gene editing efficiency, elimination of wrong sequences in DNA samples, screening of gene editing individuals and detection and screening of InDel and SNP sites in the samples, and the InDel and SNP sites are converted into molecular markers for molecular assisted breeding.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to molecular biology, genetic engineering and biotechnology applications, in particular to a method and application for enhancing the mismatch cutting ability of T7EI-like nuclease. Background technique [0002] The differences between species and individuals are determined by the differences in their genomes and gene sequences. Rapid screening to determine whether there are differences between homologous sequences and the essence of the differences is an important topic in the field of molecular biology research and biotechnology. Related research methods and technologies Not only has broad application prospects in the fields of medicine and agriculture, but also has huge market value. [0003] Whether it is a homologous sequence variation produced by natural evolution, artificial mutagenesis or directed editing, the differences between sequences can be divided into two categories: insertion...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2521/327C12Q2527/125C12Q2565/125
Inventor 范三红单丽伟胡小平
Owner NORTHWEST A & F UNIV
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