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Method for identifying gene editing rice based on high-resolution melting curve

A high-resolution melting and gene editing technology, which is applied in the identification of gene-edited rice and the identification of gene-edited rice products, can solve the problems of gene-edited rice detection methods such as vacancies

Inactive Publication Date: 2021-01-05
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purpose of the present invention is to solve the problem of vacancy in gene editing rice detection method, which has the advantages of simplicity, strong specificity, high throughput, etc.

Method used

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  • Method for identifying gene editing rice based on high-resolution melting curve
  • Method for identifying gene editing rice based on high-resolution melting curve
  • Method for identifying gene editing rice based on high-resolution melting curve

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035](1) The samples include the genomic DNA of wild-type and gene-edited rice, and the test rice sample 3#, which was collected from the scientific research base of Shandong Academy of Agricultural Sciences.

[0036] (2) The PCR primer sequence list is as follows:

[0037]

[0038] (3) Sampling and DNA extraction

[0039] The rice samples to be tested were frozen and ground in liquid nitrogen (Retsch MM400 bio-shredder), and CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM NaCl, 2 EDTA) 500 μL, lysed in a 65°C constant temperature water bath (Neslab) for 30 minutes, and the subsequent DNA extraction and purification were performed according to the resin-type genomic DNA purification kit (Saibaisheng). After DNA extraction, the concentration was uniformly diluted to 50±1 ng / μL, Nanodrop ND1000). The DNA extraction method can also be carried out according to a commercially available kit.

[0040] (4) PCR reaction

[0041] Perform PCR amplification on th...

Embodiment 2

[0046] (1) Samples include genomic DNA of wild-type and gene-edited rice, and rice sample 9# for testing. The samples were taken from the scientific research base of Tianjin Academy of Agricultural Sciences (for scientific experiments, and can provide external services).

[0047] (2) The PCR primer sequence list is as follows:

[0048]

[0049] (3) Sampling and DNA extraction

[0050] The rice samples to be tested were frozen and ground in liquid nitrogen (Retsch MM400 bio-shredder), and CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM NaCl, 2 EDTA) 500 μL, lysed in a 65°C constant temperature water bath (Neslab) for 30 minutes, and the subsequent DNA extraction and purification were performed according to the resin-type genomic DNA purification kit (Saibaisheng). After DNA extraction, the concentration was uniformly diluted to 50±1 ng / μL, Nanodrop ND1000). The DNA extraction method can also be carried out according to a commercially available kit.

[00...

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Abstract

The invention discloses a method for identifying gene editing rice based on a high-resolution melting curve. According to the method, a high-resolution melting curve technology is used for analyzing mutation basic group sites of the gene editing rice. According to the technology, sequencing analysis can be carried out on a known short sequence without a fluorescence labeling primer and electrophoresis detection. The method has the advantages of high accuracy, high efficiency, high sensitivity and the like, and a feasible method is provided for qualitative identification and analysis of gene editing type rice mutation base sites.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a method for identifying gene-edited rice products, specifically a method for identifying gene-edited rice based on high-resolution melting curve technology. By designing specific primers and utilizing high Qualitative identification of gene-edited rice using resolution melting curve technique. Background technique [0002] Rice is one of the most important food crops in my country. It not only has high yield but also has very wide adaptability. The traditional single breeding method is gradually developing towards the combination of traditional breeding and molecular breeding. As a new type of technology, gene editing technology has the characteristics of simple design, strong operability (Wan Simin 2018), and wide versatility. It is currently a very important means of molecular breeding. Scientists can use gene editing technology to control Or the DNA fragments that r...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858
Inventor 于海涛赵新王燕王璐平陈锐兰青阔王永
Owner 天津市农业科学院
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