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Detection primer set of T239C, C298T, and C893A sites of ABR3 gene, kit, and applications

A technology for detection kits and detection primers, applied in DNA/RNA fragments, microbe determination/inspection, recombinant DNA technology, etc., can solve problems such as unknown pathogenic mutations, and achieve high sensitivity and good market application prospects

Active Publication Date: 2017-11-24
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are only six pathogenic loci of ZNF644 gene detection kits for early-onset high myopia detection kits (CN102732607A), but these six pathogenic loci are only found in a small number of patients. However, the pathogenic mutations in the vast majority of patients with early-onset high myopia are unknown, and more pathogenic genes need to be detected for mutations

Method used

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  • Detection primer set of T239C, C298T, and C893A sites of ABR3 gene, kit, and applications
  • Detection primer set of T239C, C298T, and C893A sites of ABR3 gene, kit, and applications
  • Detection primer set of T239C, C298T, and C893A sites of ABR3 gene, kit, and applications

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Experimental program
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Effect test

Embodiment 1

[0028] 1. Design of specific primers

[0029] According to the ARR3 gene in the NCBI database (GeneBank sequence number is Gene ID: 407; NC_000023) T239C (that is, the 202nd base from the 5' end of the sequence shown in SEQ ID NO.5 is T / C), C298T (that is, SEQ ID The 261st base from the 5' end of the sequence shown in NO.6 is C / T) and C893A (that is, the 293rd base from the 5' end of the sequence shown in SEQ ID NO.7 is C / A) sites Specific primers were designed for the upstream and downstream sequences, and the designed specific primers are shown in Table 1.

[0030] Table 1 Specific primer sequences

[0031]

[0032] 2. Genomic DNA extraction

[0033] Genomic DNA was extracted from peripheral blood leukocytes of 200 normal persons and 200 early-onset high myopia patients by phenol / chloroform method or kit extraction method. Specific steps are as follows:

[0034] 2.1 Add 200μL Proteinase K (20mg / mL) solution to the 15mL centrifuge tube;

[0035] 2.2 Processing materia...

Embodiment 2

[0068] 1. Sensitivity detection

[0069] 1.1 Plasmid dilution

[0070] Dilute the mutant plasmids PMD19-T1, PMD19-T2, and PMD19-T3, wild-type plasmids PMD19-T4, and PMD19-T5 to a final concentration of 5 ng / μL, and then mix the mutant plasmids with their corresponding wild-type plasmids by volume ratio 0.2:9.8, 0.5:9.5, 1:9, 1.5:8.5, 2:8, 3:7, and 4:6 were mixed to prepare mutation abundances of 2%, 5%, 10%, 15%, and 20%. , 30% and 40% positive samples (Table 2). Using positive samples with different mutation abundances as templates, PCR amplification and sequencing were performed according to the method described in Example 1. The results are shown in Table 3. Judging from the sequencing chart and Table 3, the preliminary results show that the detection sensitivity of the detection kit (Sanger sequencing method) of the present invention is that the mutation abundance is 10%.

[0071] Plasmid information of table 2 mutant plasmid

[0072]

[0073] Table 3 Sensitivity t...

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Abstract

The invention discloses a detection primer set of T239C, C298T, and C893A sites of ABR3 gene, a kit, and applications. Based on the principle of Sanger sequencing having the characteristics of high sensitivity, stability, and accuracy, three mutation sites causing high myopia of ARR3 gene can be detected at the same time, and the kit can be used for the early screening and auxiliary diagnosis of high myopia, assists the early prevention, provides references for the treatment, and has a good market prospect.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection primer set, kit and application of ARR3 gene T239C, C298T and C893A sites. Background technique: [0002] Myopia is a common cause of visual impairment, among which, complications of high myopia (myopia above -6.00D) (glaucoma, cataract, vitreous opacity, retinal detachment, etc.) are the second leading cause of blindness; The disease rate is much higher than that of other races, and it is affected for life. What's more serious is that with the advancement of urbanization and changes in lifestyles, the incidence of myopia is still increasing year by year, especially among Asians living in economically developed areas. A large amount of evidence shows that high myopia is closely related to heredity. Familial hereditary high myopia, especially early-onset high myopia that existed before school age, is less affected by environmental factors and is mostly caused...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 张清炯肖学珊黎仕强贾小云郭向明
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV