Detection method for establishing gene editing rice based on pyrosequencing technology

A technology of pyrosequencing and gene editing, which is applied in the field of molecular biology and can solve the problems such as the vacancy of gene editing rice detection methods.

Inactive Publication Date: 2021-02-12
天津市农业科学院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problem of vacancy in gene editing r

Method used

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  • Detection method for establishing gene editing rice based on pyrosequencing technology
  • Detection method for establishing gene editing rice based on pyrosequencing technology
  • Detection method for establishing gene editing rice based on pyrosequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Samples: Test samples 1 and 2 are from the Institute of Plant Protection, Shandong Academy of Agricultural Sciences.

[0040] (2) Reagents: Plant DNA Extraction Kit (TIANGEN), DNA Marker (TaKaRa), GelRed dye, 2×GoTaq Green Master Mix (Promega), disodium oxalate tetraacetate (EDTA·Na2); trimethylol Aminomethane (Tris); Glacial acetic acid; Binding Buffer (QIAGEN); Annealing Buffer (QIAGEN); Washing Buffer (QIAGEN); Sepharose beads (QIAGEN). Specific sequence primers:

[0041] Forward primer sequence: 5'- ACCCTGTGCCACTTCAAAA -3'

[0042] Reverse primer sequence: 5'- GCTTTCTGACTGGTGACAGGTATT -3'

[0043] Sequencing primer sequence: 5'- TGTGGTGTTCCGCTG -3'

[0044] (3) Pyrosequencing PCR amplification system: a total volume of 50 μL, including 25 μL of 2×PCR Master Mix, 1 μL of upstream and downstream primers (10 μmol / L), 2 μL of DNA template (25 ng / μL), Make up to 50 µL with sterile water.

[0045] (4) Pyrosequencing PCR reaction amplification program: pre-denatur...

Embodiment 2

[0049] (1) Gene-edited rice and wild-type rice DNA were uniformly mixed into 6 concentration gradients at the ratio of 100%, 80%, 50%, 25%, 15%, and 0%, and blind samples S7 and S8.

[0050] (2) Reagents: Plant DNA Extraction Kit (TIANGEN), DNA Marker (TaKaRa), GelRed dye, 2×GoTaq Green Master Mix (Promega), disodium oxalate tetraacetate (EDTA·Na2); trimethylol Aminomethane (Tris); Glacial acetic acid; Binding Buffer (QIAGEN); Annealing Buffer (QIAGEN); Washing Buffer (QIAGEN); Sepharose beads (QIAGEN).

[0051] Specific sequence primers:

[0052] Forward primer sequence: 5'- ACCCTGTGCCACTTCAAAA -3'

[0053] Reverse primer sequence: 5'- GCTTTCTGACTGGTGACAGGTATT -3'

[0054] Sequencing primer sequence: 5'- TGTGGTGTTCCGCTG -3'

[0055] (3) Pyrosequencing PCR amplification system: a total volume of 50 μL, including 25 μL of 2×PCR Master Mix, 1 μL of upstream and downstream primers (10 μmol / L), 2 μL of DNA template (25 ng / μL), Make up to 50 µL with sterile water.

[0056] (4)...

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PUM

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Abstract

The invention provides a gene editing rice detection method established based on a pyrosequencing technology. The detection principle of the method is as follows, a transgenic biological system is catalyzed by four enzymes (ATP sulfatase, DNA polymerase, adenosine triphosphate diphosphatase and luciferase) together so that the transgenic biological system generates an enzyme cascade chemical reaction. According to the technology, sequencing analysis can be carried out on the known short sequence without a fluorescence labeling primer and electrophoresis detection. Due to the fact that most ofgene editing is insertion, deletion and mutation of a single gene, the technology can be used for rapidly detecting the gene editing rice. The detection method disclosed by the invention has the advantages of relatively good accuracy, high efficiency, sensitivity and the like, and a feasible method is provided for qualitative and quantitative detection and analysis of the gene editing type rice editing site.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method for gene-edited rice products, specifically a detection method for gene-edited rice established based on pyrosequencing technology, by designing specific primers and using pyrosequencing technology A method for qualitative and quantitative detection of gene-edited rice. Background technique [0002] Gene editing technology mainly uses sequence-specific nucleases to specifically cut DNA target sites to cause double-strand breaks, and induce DNA damage repair mechanisms to perform directional editing on the genome. At present, the CRISPR / Cas9 technology composed of Cas9 protein and guide RNA (gRNA) is widely used. In the process of target site editing, this method is simple and efficient, and has been widely used in various crops. The emergence of gene editing technology provides a new research idea for organism-specific improvement. At present, this te...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6869C12N15/11
CPCC12Q1/6895C12Q1/6869C12Q2600/156C12Q2600/13
Inventor 尉万聪尚宏丽赵新解美霞于海涛陈锐兰青阔王永
Owner 天津市农业科学院
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