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Human CRYBB1 (Crystallin Beta B1) gene mutation and application thereof

A nucleic acid and kit technology, applied in the field of human CRYBB1 gene mutation and its application

Active Publication Date: 2014-08-13
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no human CRYBB1 gene Q227X mutation has been reported to cause hereditary cataract disease

Method used

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  • Human CRYBB1 (Crystallin Beta B1) gene mutation and application thereof
  • Human CRYBB1 (Crystallin Beta B1) gene mutation and application thereof
  • Human CRYBB1 (Crystallin Beta B1) gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Extraction and sequencing of the genome of embodiment 1

[0028] Genomic DNA was extracted from human blood by conventional phenol-chloroform method for conventional PCR amplification.

[0029] The primer sequences are:

[0030] Forward primer: 5-CTGGTCTGGCAGGGGTCTGGGTG-3

[0031] Reverse primer: 5-TGAAGAAGGGTTGGGGCAAGGTA-3

[0032] reaction system:

[0033]

[0034]

[0035] Amplification conditions:

[0036] Pre-denatured at 95°C for 5 minutes, followed by 35 cycles of amplification (95°C for 45 seconds, 60°C for 30 seconds, 72°C for 30 seconds), and finally 72°C for 10 minutes to amplify a 300bp product for direct sequencing.

Embodiment 2

[0037] The detection of embodiment 2 mutation

[0038] The peripheral blood of the research subjects was collected and their genomic DNA was extracted. Genome-wide linkage analysis in a family with hereditary cataract to locate candidate regions. The base sequence of the SNP site of the CRYBB1 gene was obtained by sequencing technology. Combining the sequencing results (the sequencing method is carried out according to Example 1) and the analysis of bioinformatics, through the case-control analysis in the family and the SNP screening of the large sample control, it is determined that the mutation of the CRYBB1 gene is linked with hereditary cataract.

[0039] The family members in the following table all agreed to participate in this study with informed consent. All selected members received eye examinations at the Department of Ophthalmology, Zhongnan Hospital of Wuhan University, and hereditary cataract was diagnosed or ruled out.

[0040] individual number

[0041...

Embodiment 3

[0043] Embodiment 3 detection kit

[0044] Prepare a molecular diagnostic kit for hereditary cataract, which contains the following primers that can amplify the 18458 mutation of the nucleic acid sequence (SEQ ID NO.1):

[0045] Forward primer: 5-CTGGTCTGGCAGGGGTCTGGGTG-3

[0046] Reverse primer: 5-TGAAGAAGGGTTGGGGCAAGGTA-3

[0047] The mutation at position 18458 can be easily detected by high-resolution melting curve analysis of the amplification products of patients and controls.

[0048] In addition, the present invention also provides a molecular diagnostic kit using probe detection, including the probe for detecting the specific site, the sequence of which is shown in SEQ ID No.5. Since the unmutated sequence cannot hybridize with the probe, whether there is a gene mutation can be obtained through hybridization.

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PUM

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Abstract

The invention relates to human CRYBB1 (Crystallin Beta B1) gene mutation and relevancy thereof to cataract, provides a method for detecting the human CRYBB1 gene mutation, and provides a related detection kit. In the method, whether C (Cytosine)-to-T (Thymine) mutation exists is detected by determining nucleotide at a 18458th site in a sequence shown as SEQ ID NO: 1. Research finds that C-to-T single nucleotide polymorphism exists in the nucleotide at the 18458th site in the sequence shown as the SEQ ID NO: 1, in the human CRYBB1 gene; and when the C-to-T mutation occurs, Q227X in an amino acid sequence SEQ ID NO: 2 of beta B1 crystallin is caused, and truncated beta B1 crystallin protein is formed and is related to morbidity of genetic cataract. The method can be used for aided diagnosis of the cataract for finding out a potential carrier of the genetic cataract, so that a basis is provided for the prevention of the disease.

Description

technical field [0001] The present invention relates to a gene mutation of human CRYBB1 and a method for analyzing the mutation of human CRYBB1 gene, as well as the use of the mutation in assisting diagnosis in cataract. Background technique [0002] Crystallin was first described in 1893 by Find[ C.T., Untersuchungen der. Protein substanzen in den lichtbrechenden Medien des Auges. Z. Physiol. Chem. 1893; 18:61-106]. It is the main structural protein of the vertebrate lens, accounting for 90% of the total lens protein. Crystallin has a special spatial structure, which plays a very important role in maintaining the transparency of the lens. Crystallins can be divided into α-, β-, γ-crystallins according to molecular weight from large to small. [0003] β-crystallin (Beta crystallin, CRYB) can be divided into acidic protein (βA) and basic protein (βB) two subgroups, each subgroup is encoded by three genes (CRYBA1, 2, 3; CRYBB1, 2 , 3) [Jochen Graw. Genetics of crystalli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 严明郑芳饶艳杨娜周新熊陈岭
Owner WUHAN UNIV
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