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Dual-fluorescence plasmid, dual-fluorescence plasmid-Based method for analyzing deoxyribonucleic acid (DNA) mismatch repair functional activity in living cell and application thereof

An analysis method and mismatch repair technology, applied in the field of biological research, can solve the problems of poor calibration accuracy and achieve high accuracy, high sensitivity, and simple methods

Inactive Publication Date: 2011-07-13
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to construct a pair of dual fluorescent plasmids to solve the problem of poor verification accuracy at the single cell level produced by co-transfection of single-color fluorescent plasmids in the prior art

Method used

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  • Dual-fluorescence plasmid, dual-fluorescence plasmid-Based method for analyzing deoxyribonucleic acid (DNA) mismatch repair functional activity in living cell and application thereof
  • Dual-fluorescence plasmid, dual-fluorescence plasmid-Based method for analyzing deoxyribonucleic acid (DNA) mismatch repair functional activity in living cell and application thereof
  • Dual-fluorescence plasmid, dual-fluorescence plasmid-Based method for analyzing deoxyribonucleic acid (DNA) mismatch repair functional activity in living cell and application thereof

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Embodiment 1

[0063] Operate according to the analytical method for detecting DNA mismatch repair functional activity in living cells provided by the present invention, and the specific steps are as follows:

[0064] 1. Construction of P1 plasmid:

[0065] (1) PCR amplification to obtain EGFP fragments: use the pGEM-EGFP plasmid obtained by the preparation method provided in this article as a template (about 50ng), add 10×Thermolpol buffer (PlusMg 2+ ) 10 μL (purchased from NEB Company, New England Biolabs), dNTP mixture 2 μL (10 mM / L, purchased from NEB Company), sense-primer: 5'-CCGCTCGAGTCATTTTATGTTTCAGGTTC and anti-sense primer: 5'-CTAGCTAGCTTCTGCAGT CGACGGTAC-3' 6 μL each (the above primers were synthesized by Dalian Bao Bio, primer grade: PAGE, primer working concentration: 5 μM), so that the final concentration in the 100 μL PCR reaction system was 0.3 μM, and then 1 μL of Deep ventDNA polymerase (2U / μL , NEB), carry out PCR reaction to obtain EGFP fragment (hereinafter referred to ...

Embodiment 2

[0084] Operate according to the analytical method for detecting the functional activity of DNA mismatch repair in living cells provided by the present invention, and the specific steps are as follows:

[0085] 1. Construction of the P1 plasmid: PCR amplification to obtain the IRES2-Dsred2 fragment: (1) Using the pIRES2-Dsred2 plasmid (purchased from Clontech) as a template (about 50 ng), add 10 μL of 10×Pfu buffer (purchased from Bi Yuntian Company), dNTP mixture 2 μL (10 mM / L, purchased from NEB Company), sense-primer: 5-CATGATATCAGCTTCGAATTCTGCAGTC-3′anti-sense primer 5’ACATGCATGCATACATTGATGAGTTTGG-3′ each 6 μL (the above primers were synthesized by Shanghai Sangong Company , primer level: PAGE, the concentration is 5 μM), so that the final concentration in the 100 μL PCR reaction system is 0.3 μM, and then add 0.5 μL of Pfu DNA polymerase (purchased from Biyuntian Company, 5U / μL) to carry out the PCR reaction To obtain the IRES2-Dsred2 fragment, the IRES2-Dsred2 fragment co...

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Abstract

The invention discloses a dual-fluorescence plasmid carrying enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) genes, wherein the EGFP and RFP genes are positioned in the same reading frame. The constructed dual-fluorescence plasmid can realize co-expression of two fluorescence proteins by placing two fluorescence protein genes in the same reading frame. Meanwhile, the dual-fluorescence plasmid is applied to deoxyribonucleic acid (DNA) mismatch repair functional activity analysis in a living cell to improve the verification accuracy at unicellular level, and the method is simple and has high sensitivity of indicating parameters and high accuracy. Therefore, the invention provides a novel effective detecting method for DNA mismatch repair functional activity detection in the living cell. The invention also provides application of the dual-fluorescence plasmid in preparing reagent for detecting the DNA mismatch repair functional activity in the living cell.

Description

technical field [0001] The invention relates to the field of biological research, in particular to a dual fluorescent plasmid and its application, and an analysis method for detecting DNA mismatch repair function activity in living cells based on the dual fluorescent plasmid. Background technique [0002] At present, there are three traditional methods for analyzing the functional activity of mismatch repair (MMR) in organisms: the analysis method using bacteriophage M13mP2 and its derivatives as materials, the analysis method using oligonucleotides as materials, and the method based on yeast MMR function. Analytical method. These three MMR functional analysis models all use cell nucleus extracts to evaluate MMR functional activity. Not easy to count and other shortcomings. Therefore, these three methods are mainly used in the analysis of MMR functional activity in specific organisms, and it is difficult to obtain practical application and promotion in the carcinogenic mec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12Q1/68
Inventor 董巧香黄长江陈元红任湘鹏白承连葛红山
Owner WENZHOU MEDICAL UNIV
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