Screening method of tumor related gene mutation sites

A tumor-related, site-based technology, applied in genetic engineering, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effect of bedside detection, high accuracy, easy wide application and rapid promotion

Pending Publication Date: 2019-11-01
GUANGZHOU PLUSLIFE TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of CRISPR / Cas9 also has some restrictions. First, a relatively conserved PAM sequence (NGG) needs to exist near the region to be edited. Secondly, the guide RNA should be complementary to the sequence upstream of the PAM. The gRNA that specifically targets the target gene is the key technology for CRISPR / Cas12a gene knockout, etc.
At present, there is no report on the detection method of tumor-associated mutation gene based on CRISPR / Cas12a system

Method used

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  • Screening method of tumor related gene mutation sites
  • Screening method of tumor related gene mutation sites
  • Screening method of tumor related gene mutation sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Detection of tumor-related gene mutation sites based on CRISPR / Cas12a system

[0025] 1. CRISPR / Cas12a gene cloning and protein expression

[0026] The Cas12a protein gene derived from Lachnospiraceae bacterium is used, and the codon is optimized to make the gene more suitable for expression in mammalian cells. The optimized Cas12a protein gene was cloned into the pET28a plasmid with a 6-His histidine tag to facilitate protein purification and expression. The Cas12a protein recombinant expression vector was transformed, and the expression strain was BL21star (DE3).

[0027] After the Cas12a protein recombinant expression vector was transformed, protein expression, SDS-PAGE detection and gel column purification were performed, and the obtained purified Cas12a protein was stored at -80°C.

[0028] The specific protein expression conditions are as follows: when the OD600 of the culture solution is 0.6, 0.5 mMIPTG is added and cultivated for 4 hours. Bacteria w...

Embodiment 2

[0058] Example 2 Simultaneous detection of tumor-associated multi-gene mutation sites based on CRISPR / Cas12a system

[0059] In order to realize the simultaneous detection of multiple tumor-related gene mutation sites and facilitate the application of rapid multi-site screening, we have developed a multiple detection system for the above-mentioned tumor-related gene mutation targets. Perform analysis and design analysis of corresponding gRNA sequences, according to the similarity of these sequences, GC content, base homogeneity, whether there is a secondary hairpin structure, whether there is cross-reaction in the same reaction, etc., the reaction system and gRNA combination method optimize. The scheme of the present invention can realize simultaneous detection of multiple sites for 35 target sites shown in SEQ ID NO.1-35. This example presents the experiment and results of a gRNA combination scheme for the detection of multiple gene mutation sites in tumors.

[0060] 1. Tem...

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Abstract

The invention discloses a screening method of tumor related gene mutation sites. The technique integrates advantages of gRNA targeting recognition of RNA of tumor relevant mutation genetic transcription products and the characteristic that a targeting RNA sequence is detected by a CRISPR / Cas12a compound, a compound can cut report RNA having a detection marker and detectable signals are released, aCRISPR / Cas12a system is applied to detection of tumor relevant mutation genes, high-specificity high-sensitivity high-precision molecular detection on tumor related gene mutation sites at room temperature can be realized, multiple-site simultaneous detection can also be realized, and the screening method has important significance on detecting and screening of tumors. Besides, the scheme is low in detection cost and convenient and quick to operate, and has very good application prospects.

Description

technical field [0001] The invention belongs to the technical field of molecular detection and diagnosis. More specifically, it relates to a method for screening tumor-related gene mutation sites. Background technique [0002] Tumor is the biggest disease that threatens human health today. In China, tumor has surpassed coronary heart disease and stroke to become the number one cause of death. Targeted drugs aim at tumor-specific sites, and by maintaining a high local concentration, they can increase the specific lethality against tumors, have little effect on normal tissue cells, and avoid the side effects of chemotherapy. There is a correlation between targeted drugs and tumor gene mutations. Before using targeted drugs for treatment, it is necessary to confirm the genetic mutation sites of the tumor to guide, select targeted drugs, and predict whether the drug is effective. [0003] At present, the detection techniques of tumor-related gene mutation sites mainly include ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/113
CPCC12Q1/6886C12Q1/6858C12N15/113C12Q2600/156C12Q2600/16C12N2310/10C12N2310/20C12Q2537/143C12Q2521/301C12Q2563/107
Inventor 陈翀刘华勇胡洋
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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