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Method for detecting effect of 3' untranslated region on mRNA translation efficiency

An efficient and functional technology, applied in the field of research on the effect of 3'UTR on mRNA translation efficiency, can solve the problems of dynamic quantitative analysis of mRNA transcripts and protein production, etc.

Inactive Publication Date: 2016-04-13
RES INST OF SUN YAT SEN UNIV & SHENZHEN
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Problems solved by technology

The above-mentioned methods have their limitations, and they cannot perform dynamic quantitative analysis on mRNA transcripts and protein production at the same time, while the regulation of gene expression is a dynamic process involving DNA transcription and mRNA translation, which can only be determined by the final protein production or The number of ribosomes bound to mRNA does not reflect the dynamic process of 3'UTR length changes on translation efficiency

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  • Method for detecting effect of 3' untranslated region on mRNA translation efficiency
  • Method for detecting effect of 3' untranslated region on mRNA translation efficiency
  • Method for detecting effect of 3' untranslated region on mRNA translation efficiency

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[0014] In order to better illustrate the purpose, technical solutions and beneficial effects of the present invention, the following will further illustrate the present invention with the construction process of constructing the pmCS2 dual fluorescent reporter carrier and specific research examples in conjunction with the accompanying drawings.

[0015] 1. Construction process of dual fluorescent reporter gene carrier pmCS2

[0016] During the construction process, all synthetic primers were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; except for the reagents whose sources were indicated, the rest were domestic analytical reagents.

[0017] T4DNALigase, T4PNK, dephosphorylase, restriction endonuclease BbsI and BglII are products of NEB Company; LATaqpolymerase, restriction endonuclease EcoRI and SalI were purchased from Takara Company; fluorescent dye DFHBI-1T was purchased from Lucerna Company.

[0018] The construction method of the dual fluorescent reporter ge...

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Abstract

The invention relates to a method for detecting effect of a 3' untranslated region on mRNA translation efficiency by quantifying transcripts and protein yield in cells at the same time through a dual-fluorescence reporter system, and belongs to the field of molecular biology. The dual-fluorescence reporter gene vector is created, a coding fluorescent RNA molecular sequence is inserted behind a fluorescent protein coding region, then the searched 3' UTR sequence is inserted between the two, a recombinant vector performs transient transfection of cells, quantitative fluorescence analysis is carried out on transcript and protein expression quantity in an imaging culture medium at the same time, and the effect of the 3' UTR on the translation efficiency is judged by comparing fluorescence intensities.

Description

technical field [0001] The present invention relates to a method for studying the effect of 3'UTR on mRNA translation efficiency in cells. Background technique [0002] Alternative polyadenylation (alternative polyadenylation, APA) plays an important role in the regulation of gene expression. APA located downstream of the stop codon can produce mRNA isoforms with different 3'UTR lengths, which can lead to the inclusion or deletion of cis-regulatory elements, including RNA-binding protein (RNAbindingprotein ,RBP) binding sites and miRNA binding sites, etc., thereby affecting the transport, stability, translation efficiency, etc. of mRNA, and then affecting the phenotype. Recent studies have found that genome-wide 3'UTR transitions exist in different physiological and cellular states, including tumorigenesis and metastasis, animal development, and immune responses. Therefore, studying the effect of APA on the regulation of gene expression is of great significance for reveali...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85G01N33/68
Inventor 付永贵
Owner RES INST OF SUN YAT SEN UNIV & SHENZHEN
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