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Detection kit of disease-causing gene CRYGD of crystalline congenital cataract

A congenital cataract and detection kit technology, applied in the field of genetic detection kits, to achieve the effect of reducing the missed detection rate

Active Publication Date: 2013-04-17
山西省眼科医院
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007]In the known report, C-->A occurs at the 70th base in the second exon of the crystal-like congenital cataract gene CRYGD gene Substitution changes lead to the change of proline at position 23 to threonine (P23T), which changes the structure of γ-crystallin and causes cataracts, but there is no better way to locate and detect it

Method used

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  • Detection kit of disease-causing gene CRYGD of crystalline congenital cataract
  • Detection kit of disease-causing gene CRYGD of crystalline congenital cataract
  • Detection kit of disease-causing gene CRYGD of crystalline congenital cataract

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Implementation 1 Chromosomal mapping of disease-causing genes

[0026] 1. Genomic DNA extraction

[0027] Whole blood genomic DNA was extracted by standard saturated phenol / chloroform extraction.

[0028] 2. Linkage analysis

[0029] (1) Selection of microsatellite marker sites: 22 pairs of polymorphic microsatellite genetic markers within 5Mb distance from 17 known ADCC-causing genes were selected, and the physical parameters of each ADCC candidate gene and its surrounding microsatellite markers were selected. The distances are shown in Table 1.

[0030] Table 1: Physical distance of 17 known ADCC pathogenic genes and their nearby microsatellite markers

[0031]

[0032] (2) Amplification of microsatellite markers:

[0033] The multiplex PCR reaction adopts the Touch Down PCR program, with a total of two-phase cycles: pre-denaturation at 95°C for 5 rain, the first phase cycle at 94°C for 30 s, the initial annealing temperature at 63°C for 45 s, and a decreas...

Embodiment 2

[0043] Example 2 Candidate Gene Mutation Detection

[0044] 1. Sequencing analysis of candidate gene mutations

[0045] PCR reaction amplification: According to the entire coding sequence of CRYGC gene and CRYGD gene provided in Table 3, the junction region between exon and intron, and the 5' and 3' untranslated regions (5'UTR and 3'UTR), the primer sequences used Primer 3 was designed by itself and synthesized by Dalian Bao Biological Company.

[0046] The PCR reaction system includes 150 ng of genomic DNA; 2ul of 10×PCR buffer; 0.5ul of dNTP mixture; 0.5ul of upstream and downstream primers (both 10 umol / L); 0.5ul of Taq DNA polymerase (2 U / ul); Water 14ul. PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 10 min, a total of 32 cycles, and storage at 4°C. PCR amplification products were electrophoresed on 1% agarose gel at 120 V constant voltage for 10 min. After purification ...

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Abstract

The invention discloses a detection kit of a disease-causing gene CRYGD of a crystalline congenital cataract, and relates to a gene detection case. The detection kit of the disease-causing gene CRYGD of crystalline congenital cataract comprises an AS-PCR amplification detection reagent. The AS-PCR amplification detection reagent comprises 2 microlitre of 10*PCR buffer, 0.5 microlitre of dNTP mixture, 0.5 microlitre of 10 micromol / L AS-PCR special upstream primer and downstream primer and inner reference upstream primer and downstream primer respectively, 0.5 microlitre of 2U / microlitre TaqDNA polymerase and 13 microlitre of deionized water. CRYGD gene P23T point mutation is directly detected by DNA group extracted in patient blood, so that crystalline congenital cataract can be accurately diagnosed, so that the omission ratio is reduced, and the crystalline congenital cataract can be identified with other congenital cataracts.

Description

technical field [0001] The invention relates to a gene detection box, in particular to a detection kit for crystal-like congenital cataract pathogenic gene CRYGD. Background technique [0002] Congenital cataracts are different degrees and forms of lens opacity caused by abnormal lens metabolism during fetal development, which not only blurs the retinal image, but also prevents the development of the visual pathway. It is currently the main cause of low vision and blindness in children. The incidence rate of this disease is about 0.5%, accounting for the second place among children's blinding eye diseases. One-third of the pathogenic factors of congenital cataract are related to heredity, and the modes of inheritance include autosomal dominant inheritance, autosomal recessive inheritance and x-linked inheritance. Disease-causing gene screening is an important step in exploring the molecular pathogenesis of diseases. [0003] The current study found that 39 loci and 17 gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张素华张哲贾亚丁张晓慧
Owner 山西省眼科医院
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