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Application of abca3 gene in the preparation of congenital ccmc diagnostic products

A technology for congenital cataracts and products, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of limited understanding of disease-causing genes

Active Publication Date: 2016-06-01
SHANDONG EYE INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the understanding of the causative genes of CCMC is still limited, and the 7 known genes can only explain the pathogenesis of less than 30% of patients so far

Method used

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  • Application of abca3 gene in the preparation of congenital ccmc diagnostic products
  • Application of abca3 gene in the preparation of congenital ccmc diagnostic products
  • Application of abca3 gene in the preparation of congenital ccmc diagnostic products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Screen the mutation site of ABCA3 gene from congenital CCMC family

[0065] 1. Extraction of peripheral blood genomic DNA:

[0066] In compliance with the relevant national policies and regulations, and on the basis of the consent of the sampling subject, draw 2-5ml of peripheral venous blood from the first family member, put it into an EDTA anticoagulant tube, and store it at -80°C for later use; the frozen EDTA anticoagulant blood After melting at room temperature, put 500 μL into a centrifuge tube, add an equal volume of TE (pH 8.0), mix well, centrifuge at 10,000 rpm for 10 minutes at 4°C, and discard the supernatant.

[0067] Add 180 μLTE, 20 μL SDS (10%), 8 μL proteinase K (10 mg / ml), mix well, and place in a 37°C water bath overnight. Remove the sample from the water bath and briefly centrifuge to pellet the sample. Add an equal volume of Tris-saturated phenol (about 300 μL) to the reaction tube, mix thoroughly, centrifuge at 10,000 rpm for 10 min...

Embodiment 2

[0122] Example 2: Screening the mutation site of ABCA3 gene from another congenital CCMC family

[0123] 1. Extraction of genomic DNA from peripheral blood: the conventional phenol-chloroform method described in Example 1 was used to extract genomic DNA from the peripheral blood of each member of the family.

[0124] 2. PCR amplification target fragment: reaction conditions and reaction system:

[0125] (1) PCR reaction conditions: 94°C for 3min; 94°C for 40sec, 55±3°C for 40se, 72°C for 60sec, 30-35cycle; 72°C for 10min.

[0126] (2) Reaction system: (TAKARALATaqpolymerase)

[0127]

[0128] The reaction system was used to amplify each patient's genomic DNA template and 19 pairs of primers for the ABCA3 gene.

[0129] 3. Sequencing of PCR products: The genomic DNA templates of the patients in the family and the amplification products of 19 pairs of primers of the ABCA3 gene were sequenced using the conventional Sanger sequencing method. A heterozygous mutation occurred ...

Embodiment 3

[0130] Example 3: Screening the mutation site of ABCA3 gene from another 2 patients with congenital sporadic CCMC

[0131] Collection of sporadic patients with CCMC: A total of 2 sporadic patients with definite diagnosis of CCMC were collected in Shandong Eye Institute and Qingdao Eye Hospital.

[0132] Genomic DNA extraction from peripheral blood:

[0133] Take 2-5ml of peripheral venous blood from 2 patients with sporadic CCMC, put them into EDTA anticoagulant tubes, and freeze them at -80°C for later use; after thawing the frozen EDTA anticoagulated blood at room temperature, put 500 μL into centrifuge tubes, and use them for implementation. The conventional phenol-chloroform method described in Example 1 was used to extract genomic DNA from the peripheral blood of each member of the family.

[0134] PCR amplification target fragment: reaction conditions and reaction system:

[0135] (1) PCR reaction conditions: 94°C for 3min; 94°C for 40sec, 55±3°C for 40se, 72°C for 60s...

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Abstract

The invention aims at providing an application of an ABCA3 gene in preparation of a diagnostic product for detecting CCMC (congenital cataract microcornea complex syndrome). A new application of the ABCA3 gene is provided, and sequentially, an effective way for performing CCMC disease genetic diagnosis, prenatal gene screening and genetic counseling is provided; and the application effect shows that SNP (Single Nucleotide Polymorphism) locus and detection primer of the gene provided by the invention can be effectively used for ABCA3 gene mutation locus rapid detection on clinic patients and chorionic villus or amniotic fluid.

Description

technical field [0001] The invention belongs to the related technical field of gene diagnosis and prenatal gene diagnosis, and specifically relates to the application of ATP junction box (ABC) transporter A3 (ABCA3) in the preparation and detection of congenital cataract-microcorneal syndrome (CCMC) gene diagnosis products. Background of the invention [0002] Congenital cataract is a common eye disease that causes low vision and blindness in children. The incidence rate is about 0.01% to 0.06%, and about 50% of them are related to genetics. The most common mode of inheritance is autosomal dominant inheritance. Congenital cataract can occur independently or as a concomitant symptom of ocular or systemic syndromes, among which 12%-18% of patients with congenital cataract often have symptoms of microcornea. Cataract-microcornea syndrome (Cataract-microcorneayndrome, CCMC, OMIM116200) is a congenital dysplasia eye disease, often involving both eyes, manifested as different phen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 陈鹏代云海谢立信王晔肖晶晶张清岩管李萍王俊
Owner SHANDONG EYE INST
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