Method for detecting trinucleotide repeats in mammalian genome and its application

A repetitive sequence and trinucleotide technology, applied in the field of nucleotide detection, can solve problems such as inability to perform specific analysis

Active Publication Date: 2018-03-02
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current research methods, even gene-wide association studies (GWAS) cannot perform specific analysis on the role of each gene in such polygenic diseases, especially the genes where the trinucleotide repeats are located, so it is necessary to develop A method for the systematic study of trinucleotide repeat sequences from a genome-wide perspective

Method used

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  • Method for detecting trinucleotide repeats in mammalian genome and its application
  • Method for detecting trinucleotide repeats in mammalian genome and its application
  • Method for detecting trinucleotide repeats in mammalian genome and its application

Examples

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specific example

[0064] As a specific example, the method includes:

[0065] (1) incubating the special sequence binder and the DNA under conditions suitable for the binding of the special sequence binder to the DNA;

[0066] (2) Isolate the bound specific sequence binder-DNA complex;

[0067] (3) DNA obtained by separating and purifying the special sequence binder-DNA complex obtained in step (2); and

[0068] (4) Sequencing the DNA separated and purified in step (3).

[0069] Herein, special sequences include, but are not limited to, trinucleotide repeat sequences, including (GCT)n, (CTG)n, (TGC)n, (AGC)n, (GCA)n, (CAG)n, (GGC) n, (GCG)n, (CGG)n, (GCC)n, (CCG)n, (CGC)n, (GAA)n, (AAG)n, (AGA)n, (TTC)n, (TCT)n n and (CTT)n, where n is an integer greater than or equal to 3. In a specific embodiment, n≤100, for example, n is an integer between 3-50, 3-30, and 3-20.

[0070] Herein, the specific sequence combination includes zinc finger protein or transcriptional activator-like effector, and...

Embodiment 1

[0155] Example 1: Specific binding of recombinant zinc finger protein to target DNA

[0156] We designed a specific recognition GCT trinucleotide repeat sequence (GCT) based on the zif268 zinc finger protein 3 zinc finger protein ZFP GCT (SEQ ID NO:1), which is connected by three tandem zinc finger domains, in which the specific amino acid residues that recognize the helix are in figure 1 listed in A. We constructed the artificially designed recombinant gene on the E.coli expression vector, and purified it with NTA-Ni column after inducing expression.

[0157] We used the same method to prepare the zinc finger protein ZFP GGC (SEQ ID NO:2) and ZFP GAA (SEQ ID NO: 3).

[0158] At the same time, we constructed the recombinant gene into a eukaryotic cell expression vector, transfected it into HeLa cells through liposomes, and verified the localization of the protein in eukaryotic cells by immunofluorescence. By co-staining with DAPI dye, we can see the ZFP GCT The -flag fu...

Embodiment 2

[0162] Example 2: ZFPs GCT Binds specifically to CTG, TGC, GCT, AGC, GCA and CAG repeats

[0163] We used EMSA experiments and in vitro ZIP experiments (ie, the aforementioned in vitro DNA immunoprecipitation) to prove that ZFP GCT Protein specific binding to GCT repeats. Such as figure 1 As shown in F, there are multiple CAG repeats in the HTT gene, each of which has a ZFP GCT The protein can bind three GCT repeats, so HTT genomic DNA can be read by multiple ZFPs GCT protein binding. When ZFP GCT When binding to a sequence containing ≥ 3 repeated GCTs, the reading frame is shifted, and it can also specifically bind to CTG, TGC and AGC, GCA, CAG repeat sequences of the complementary chain. At the same time, the method of semi-quantitative PCR was used to detect the DNA enriched by ZIP in vitro, such as figure 1 As shown in G, compared with the non-specifically enriched GAPDH gene fragment, the specifically enriched SCA1 gene fragment can be significantly amplified.

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Abstract

The invention relates to a method for detecting a trinucleotide repeated sequence of a mammal genome and an application thereof, and specifically, the invention relates to a method for detecting the trinucleotide repeated sequence of the mammal genome by using C2H2 zinc finger protein. The invention also relates to the specific C2H2 zinc finger protein, a coding sequence of the specific C2H2 zinc finger protein, polynucleotide constructs containing the coding sequence of the specific C2H2 zinc finger protein, cells, and an application of the C2H2 zinc finger protein, the coding sequence, the polynucleotide constructs and the cells.

Description

technical field [0001] The invention relates to the field of nucleotide detection, in particular to a method for detecting a mammalian genome trinucleotide repeat sequence and an application thereof. Background technique [0002] Trinucleotide repeat (trinucleotide repeat, TNR), an expansion of a trinucleotide sequence ubiquitous in mammals, which affects normal gene function through gain or loss mutations at the protein or RNA level, Causes degenerative diseases related to the neuro-muscular system. This instability of trinucleotide repeats appears to change dynamically during tissue and its passage, and longer repeats are more likely to undergo mutations that increase repeat number than shorter ones. At the same time, because the length of the repeat sequence is related to the severity of the disease and the age of onset of the patient, most of the diseases caused by trinucleotide repeats show the characteristics of genetic early onset. With changes in development, appea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C07K14/47C12N15/12C12N15/70C12N1/21
CPCC07K14/47C12Q1/6806C12Q1/6869C12Q2522/101C12Q2525/151
Inventor 胡荣贵徐星星傅兴陈侃于涛
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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