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Beta-thalassemia genotyping method

A technology for thalassemia and genotyping, applied in biochemical equipment and methods, DNA/RNA fragments, microbial determination/inspection, etc., to achieve the effect of low detection cost, high sensitivity, and convenient operation

Pending Publication Date: 2019-09-03
GUANGZHOU PLUSLIFE TECH CO LTD
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Problems solved by technology

The applicant found that there is no relevant report on the detection method of β-globin gene (HBB) mutation based on CRISPR / Cas12a system

Method used

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Embodiment 1

[0026] Example 1 β-thalassemia genotyping detection based on CRISPR / Cas12a system

[0027] 1. CRISPR / Cas12a gene cloning and protein expression

[0028] 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).

[0029] 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.

[0030] 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 were collecte...

Embodiment 2

[0056] Example 2 Simultaneous detection of multiple gene mutation sites in β-thalassemia based on CRISPR / Cas12a system

[0057] In order to realize the simultaneous detection of multiple β-thalassemia gene mutation sites and facilitate the application of rapid multi-site screening, we have developed a multiple detection system for the above-mentioned β-thalassemia gene mutation targets. Analyze the thalassemia gene mutation sequence, and design and analyze the corresponding gRNA sequence. System and gRNA combinations were optimized. The gRNA in Example 1 can realize multi-site simultaneous detection for the corresponding target gene. This example presents the experiment and results of a gRNA combination scheme for the detection of multi-gene mutation sites in β-thalassemia.

[0058] 1. Template preparation The preparation of templates to be detected was prepared according to step 2 in Example 1, and SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 were prepared as templates.

[00...

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Abstract

The invention discloses a beta-thalassemia genotyping method. Based on the method, a CRISPR / Cas12a-based beta thalassemia genotyping assay kit is constructed. The method has good specificity, high sensitivity and high precision for beta-thalassemia genotyping, can simultaneously detect multiple sites, and has great significance for the detection and screening of beta thalassemia. At the same time,the scheme has low detection cost, convenient and fast operation, and has broad 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 of beta thalassemia genotyping. Background technique [0002] β-thalassemia (Hemophilia) is a hemolytic anemia caused by the deletion or defect of the β-globin (HBB) gene, which inhibits the synthesis of the β-globin chain, and is mostly caused by point mutations in the β-globin gene. Those who cannot synthesize β chain at all are called β0, and those who can partially synthesize β chain (5% to 30% of normal) are called β+. β-thalassemia is one of the most common hereditary diseases in the world. According to statistics, more than 100 million people in the world carry the β-thalassemia gene. [0003] The genetic diagnosis methods for β-thalassemia mainly include: Sanger sequencing, restriction fragment length polymorphism (RFLP), reverse dot blot (RDB), and allele-specific PCR (ARMS-PCR). The Sanger sequencing method is cumber...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/113
CPCC12Q1/6883C12Q2600/156
Inventor 刘华勇陈翀黄嘉恩
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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