Kit for detecting cerebral apoplexy related TYMS gene based on CRISPR

A detection kit and gene technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of inability to detect gene mutations in clinical samples on a large scale, long detection time, low accuracy, etc. problem, to achieve the effect of high sensitivity, high accuracy and high conversion efficiency

Pending Publication Date: 2022-05-06
江苏博嘉生物医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of this, the purpose of the present invention is to solve the problems of long detection time, high cost and low accuracy in the prior art, so that it is impossible to detect gene mutations in clinical samples on a large scale, and provides a method based on CRISPR technology to detect stroke-related Whether there are rs699517 and / or rs2790 target mutation sites in the TYMS gene and a kit for detecting whether the target nucleic acid has a mutation in the target region

Method used

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  • Kit for detecting cerebral apoplexy related TYMS gene based on CRISPR
  • Kit for detecting cerebral apoplexy related TYMS gene based on CRISPR
  • Kit for detecting cerebral apoplexy related TYMS gene based on CRISPR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 Design and acquisition of crRNA targeting gene mutation site

[0049] (1) Design principles of crRNA targeting gene mutation sites

[0050] Since the CRISPR-Cas12a system is a novel targeted DNA gene editing system, in which Cas12a combines with crRNA to form a monitoring complex, the guide region of crRNA recognizes the target DNA with a complementary sequence, and Cas12a degrades the target DNA strand. Wherein the crRNA design requirement: crRNA includes protein anchor sequence and guide sequence, sequence format is: 5`-anchor sequence combined with Cas12a protein-crRNA guide sequence-3`, protein anchor sequence needs to be determined according to Cas12a protein , so that it can match and bind to the selected Cas12a protein; the guide sequence matches the fragment in the targeted DNA.

[0051] (2) Selection of crDNA sequence

[0052] The cas12a protein selected in this embodiment is FnCas12a, so the anchor sequence selected to bind to the Cas12a protein i...

Embodiment 2

[0062] Example 2 Detection kit and detection method for stroke-related TYMS gene mutation sites

[0063] 1. The composition of the kit for the detection of TYMS gene and stroke-related mutation sites

[0064] 1) 2 mutant crRNAs (obtained as shown in Example 1), the sequences of which are shown in SEQ ID NO.3-4;

[0065] Or 2 mutant crDNAs (when the kit is crDNA, the user needs to first generate RNA from the crDNA fragments under the action of T7 RNA polymerase, recover and purify the crRNA, see Example 1 for details), its sequence is as SEQ ID NO .1 ~ 2 shown.

[0066] 2) A specific fluorescent probe, in this embodiment, the fluorescent probe is specifically any one of Table 2:

[0067] Table 2 Fluorescent probe sequences

[0068]

[0069]

[0070] 3) cas12a protein, in this embodiment, Fncas12a is used.

[0071] 4) Enzyme-free water and DNase inhibitors.

[0072] 5) A primer pair for specifically amplifying rs699517 and rs2790 polymorphic sites on the TYMS gene, th...

Embodiment 3

[0084] Example 3 Detection of TYMS Gene Mutation Sites Related to Stroke

[0085] 1) Take 10-50 ng of sample DNA to be tested and add it to the amplification system shown in Table 3, wherein the primers are corresponding amplification primer pairs shown in SEQ ID NO.5-8.

[0086] Table 3 PCR amplification system

[0087]

[0088]

[0089] 2) Add the obtained amplification product to the detection mixture (the mixture contains 100-200 nM purified FnCas12a, 100-200 nM crRNA, 1-5 μL synthetic fluorescent probe, 2 μL DNase inhibitor and enzyme-free water) , incubate at 37°C for 0.5-1h in detection buffer (NEBuffer 2.1), the detection system is shown in Table 4:

[0090] Table 4 detection system

[0091]

[0092] After incubating the detection system, use a fluorescence detector to continuously measure the fluorescence value, and at the same time use wild-type crRNA to set up a negative control group, and judge whether there are corresponding molecular markers related to...

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Abstract

The invention belongs to the technical field of gene mutation detection, and discloses a CRISPR (clustered regularly interspaced short palindromic repeats)-based kit for detecting a cerebral apoplexy related TYMS gene, the kit comprises mutant crDNA (deoxyribonucleic acid) or mutant crRNA (ribonucleic acid) designed for rs699517 and rs2790 loci of the TYMS gene, and the sequence of the mutant crDNA or the mutant crRNA is shown as SEQ ID NO.1-4. By combining a CRISPR-cas12a system, whether gene mutation of the rs699517 and/or rs2790 loci of the TYMS gene exists in a sample to be detected or not can be determined, and whether the gene mutation of the rs699517 and/or rs2790 loci of the TYMS gene The kit has the advantages of high specificity, high accuracy, high detection speed and the like, and has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of gene mutation detection, in particular to a CRISPR-based kit for detecting stroke-related TYMS gene mutations. Background technique [0002] Stroke, also known as stroke, is a complex multifactorial polygenic disease caused by multiple gene-gene and gene-environment interactions. Multiple factors, including hypertension, diabetes, smoking, hyperlipidemia, and hyperhomocysteinemia, are associated with a higher risk of stroke. Hyperhomocysteinemia, in particular, has been identified as an independent, potentially modifiable risk factor for ischemic stroke in a multiethnic population. Studies have shown that hypertension, impaired fasting glucose, and hyperhomocysteinemia are considered independent risk factors for cerebrovascular disease, and the molecular basis of this risk is attributed to the folate cycle, methionine cycle, and thymidylate synthesis Abnormal function of factors in enzymes (DNA repair) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11C12Q1/686
CPCC12Q1/6883C12Q1/686C12Q2600/156C12Q2521/327C12Q2563/107C12Q2527/127
Inventor 肖以磊于福华孙汉宇刘岳飞周杰邢晓辉李中辰程诚姚杰
Owner 江苏博嘉生物医学科技有限公司
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