gRNA for targeting to HBB RNA, and HBB mutation detection method and detection kit based on C2c2

A c2c2, gene technology, applied in the direction of DNA / RNA fragments, other methods of inserting foreign genetic materials, recombinant DNA technology, etc.

Pending Publication Date: 2019-07-02
GUANGZHOU PLUSLIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] The applicant found that there is no relevant report on the detection method of β-globin gene (HBB) mutation based on CRISPR-C2c2 system

Method used

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  • gRNA for targeting to HBB RNA, and HBB mutation detection method and detection kit based on C2c2
  • gRNA for targeting to HBB RNA, and HBB mutation detection method and detection kit based on C2c2
  • gRNA for targeting to HBB RNA, and HBB mutation detection method and detection kit based on C2c2

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Experimental program
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preparation example Construction

[0118] 2. Preparation of target RNA gRNA preparation of target nucleotides:

[0119]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 T7Quick High Yield RNASynthesis kit (New England Biolabs) kit), and then using MEGAclear TranscriptionClean -up kit (Thermo Fisher) kit was used to purify RNA to obtain target nuclear RNA.

[0120] NASBA isothermal amplification

[0121] At 4°C, configure the amplification system as follows:

[0122]

[0123] The above mixed system was placed at 65°C for 2 minutes; then at 41°C f...

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Abstract

The invention provides a gRNA for specifically targeting to HBB RNA, and also provides a mutation detection method and detection kit for human beta globin gene (HBB) based on a regular interval clustered short palindrome repeat sequence (CRISPR)-C2c2 system. The invention provides the detection method, the advantages of the gRNA for targeting recognition of the HBB gene transcription product RNA (target RNA sequence) and the characteristic that a CRISPR-C2c2 complex can cut a report RNA with a detection marker and release detectable signals when the target RNA sequence is detected by the CRISPR-C2c2 complex are integrated. The CRISPR-C2c2 system is applied to detection of the mutation of the beta globin gene (HBB), and the detection method and detection kit have high sensitivity and accuracy, and are a detection method and detection kit with great commercial 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 HBB RNA, and a beta globin gene (HBB) mutation based on a clustered regularly interspaced short palindromic repeat (CRISPR) system Detection methods, detection kits. Background technique [0002] The clustered regularly interspaced short palindromic repeat system (clustered regularly interspaced shortpalindromic repeat; CRISPR-associated, CRISPR-Cas) is an important immune defense system for archaea and bacteria against virus and plasmid infection, and is used to resist foreign genetic material. Invasions, such as phage viruses and foreign plasmids. At the same time, it provides acquired immunity for bacteria: this is similar to the acquired immunity of mammals. When bacteria are invaded by viruses or foreign plasmids, they will generate corresponding "memory" to resist their re-invasion. The CRISPR / Cas system can recognize for...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/63C12N15/90C12Q1/6827
CPCC12N15/113C12N9/22C12N15/63C12N15/902C12Q1/6827C12Q2563/107C12Q2521/301
Inventor 陈翀罗镇华
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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