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Plasmid for verifying influence of human gene Intron variation on gene splicing and construction method and application thereof

A technology of gene splicing and construction method, applied in the field of genetic engineering, can solve the problems of gene splicing and no systematic verification method.

Pending Publication Date: 2021-12-21
浙江赛微思生物科技有限公司
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Problems solved by technology

[0002] With the wide application of whole-exome sequencing and whole-genome sequencing in the field of rare genetic diseases, more and more gene intron region variations have been reported, but the impact of intron region variation on gene splicing is currently There is no systematic verification method

Method used

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  • Plasmid for verifying influence of human gene Intron variation on gene splicing and construction method and application thereof
  • Plasmid for verifying influence of human gene Intron variation on gene splicing and construction method and application thereof
  • Plasmid for verifying influence of human gene Intron variation on gene splicing and construction method and application thereof

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Embodiment Construction

[0024] The present invention will be further explained below in conjunction with the embodiments and the accompanying drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0025] An alternative splicing reporter plasmid, based on the plasmid pCDNA3.1-EGFP-C (Addgene accession number 129020), divides the EGFP gene sequence into three segments and named them E1, E2, and E3, and breaks the Insert two intron sequences Intron1 and Intron2 at the point. Intron1 and Intron2 have 5' cleavage site, branch site and 3' cleavage site respectively, so as to ensure that U1snRNP can bind with 5' cleavage site to start Splicing, the U2AF factor binds to the region between the branch site and the 3' splicing site, so that the intron can form a lariat structure for splicing, and the transformed plasmid, the alternative splicing reporter plasmid, is named pCDNA3.1 -EGFP-AS.

[0026] The sequences ...

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Abstract

The invention provides a plasmid for verifying the influence of human gene Intron variation on gene splicing. An Intron of a gene to be verified is used for replacing Intron1 or Intron2 of a variable splicing reporter plasmid pCDNA3.1-EGFP-AS to form a minigene plasmid containing the Intron of the gene to be verified. The invention further provides a construction method and application of the plasmid. By forming the minigene plasmid containing the Intron of the gene to be verified and combining cell transfection and cDNA detection, the influence of the gene Intron variation on gene splicing can be embodied at the cell level and the living body level, and variable splicing practice can be detected at the transcription level.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a plasmid for verifying the influence of human gene intron variation on gene splicing and its construction method and application. Background technique [0002] With the wide application of whole-exome sequencing and whole-genome sequencing in the field of rare genetic diseases, more and more gene intron region variations have been reported, but the impact of intron region variation on gene splicing is currently There is no systematic method of verification. In recent years, studies have suggested that mutations located in introns in many disease-related genes affect the splicing process of pre-mRNA. The most common forms of splicing mutations result in aberrant splicing by creating or enhancing splice sites or creating new branch points. In specific introns, mutations can create new splice sites that serve as boundaries for new exons, allowing the newly created exo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/113C12Q1/02G01N21/64
CPCC12N15/85C12N15/65C12N15/113G01N21/6428G01N33/5005C12N2800/107C12N2800/70
Inventor 王义亭刘静梁萌萌
Owner 浙江赛微思生物科技有限公司
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