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GRNA targeted to RNA of Rett mutation gene, and detection method and detection kit for the Rett mutation gene

A technology of mutated genes and target nucleotides, applied in the field of detection kits and Rett syndrome-related mutated genes, which can solve difficult problems such as treatment

Pending Publication Date: 2019-03-01
GUANGZHOU PLUSLIFE TECH CO LTD
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Problems solved by technology

Most (80%) adults have symptoms of epilepsy, some women have improved spastic activity with age, but some have difficult to treat condition, mostly requiring medication to control
[0005] The applicant found that there is no report on the detection method of Rett syndrome-related mutation gene based on CRISPR-C2c2 system

Method used

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  • GRNA targeted to RNA of Rett mutation gene, and detection method and detection kit for the Rett mutation gene
  • GRNA targeted to RNA of Rett mutation gene, and detection method and detection kit for the Rett mutation gene
  • GRNA targeted to RNA of Rett mutation gene, and detection method and detection kit for the Rett mutation gene

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preparation example Construction

[0103] 2. Preparation of target RNA gRNA preparation of target nucleotides:

[0104] Extract target nucleotides through PCR amplification, recombinase polymerase amplification (RPA), NASBA isothermal amplification or, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification DNA amplification (HDA) and nickase amplification reaction (NEAR) to amplify target DNA. Gel separation and purification (using the MinElute gel extraction kit (Qiagen) kit), the purified dsDNA was incubated with T7 polymerase at 30°C overnight (using the HiScribe T7 Quick High Yield RNASynthesis kit (New England Biolabs) kit), and then using MEGAclear TranscriptionClean-up kit (Thermo Fisher) was used to purify RNA to obtain target nuclear RNA.

[0105] NASBA isothermal amplification

[0106] At 4°C, configure the amplification system as follows:

[0107]

[0108] The above mixed system was placed at 65°C for 2 minutes; then at 41°C for ...

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Abstract

The invention provides gRNA targeted to Rett syndrome-related mutation gene RNA and provides a detection method and a detection kit for human Rett syndrome-related mutation on the basis of a clusteredregularly interspaced short palindromic repeats (CRISPR)-C2c2 system. In the detection method, by means of advantages of the gRNA targetedly-recognizing a transcription product RNA (target RNA sequence) of the Rett syndrome-related mutation gene, when the CRISPR-C2c2 composite detects the target RNA sequence, the composite can cut the report RNA having a detection label and release a detectable signal, so that when the CRISPR-C2c2 system is applied to detection for the Rett syndrome-related mutation gene, high sensitivity and accuracy are achieved. The detection method and the detection kit have huge commercial application potential value.

Description

technical field [0001] The invention relates to the field of gene detection and gene modification, and relates to a gRNA for specifically targeting Rett syndrome-related mutant gene RNA, and a Rett syndrome based on a clustered regularly interspaced short palindromic repeat (CRISPR) system Related mutant gene detection methods, detection kits; especially a gRNA for specifically targeting Rett syndrome resistance genes and / or specific gene RNAs, and a method based on regularly interspaced clustered short palindromic repeats (CRISPR) ) Systematic Rett syndrome resistance gene and / or specific gene detection method and detection kit. Background technique [0002] CRISPR / Cas is a powerful tool for gene editing, which can precisely edit genes at a specific point. [0003] On April 13, 2017, Science magazine published an important research progress entitled "Nucleic acid detection with CRISPR-Cas13a (ie C2c2)". A team of scientists from the Broad Institute, the McGovern Institute...

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N9/22C12Q1/6883
CPCC12N9/22C12N15/113C12N15/907C12Q1/6883C12N2310/00C12Q2600/156
Inventor 陈侃罗镇华
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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