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Autosomal dominant Dnajc17 gene mutant as well as application, diagnostic kit and diagnostic gene chip thereof

A diagnostic kit and autosome technology, applied in the field of biomedicine, to achieve the effect of great clinical application value

Active Publication Date: 2020-03-24
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have shown that some other proteins in the same family as Dnajc17, such as Dnajc5 and Dnajc14, are related to the development of inner ear hair cells or the maintenance of hearing function, but there is no direct evidence to explain the relationship between Dnajc17 and deafness, or to confirm that its gene mutation is One of the genetic causes of deafness

Method used

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  • Autosomal dominant Dnajc17 gene mutant as well as application, diagnostic kit and diagnostic gene chip thereof
  • Autosomal dominant Dnajc17 gene mutant as well as application, diagnostic kit and diagnostic gene chip thereof
  • Autosomal dominant Dnajc17 gene mutant as well as application, diagnostic kit and diagnostic gene chip thereof

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Experimental program
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Effect test

Embodiment 1

[0035] By performing exome sequencing on a family with autosomal dominant non-syndromic deafness, the present invention found that there is a significant correlation between the mutation of Dnajc17 gene on human chromosome 15 (c. 401A>C, p. E134A) and the occurrence of deafness . Based on this, it was deduced that Dnajc17 could be used as a biomarker for the diagnosis of deafness.

[0036] The verification of the correlation between Dnajc17 and deafness diseases, that is, the determination of Dnajc17 as a biomarker for the diagnosis of deafness: includes the following steps:

[0037] The first step, sample preparation: clinically found a family of acquired deafness (such as figure 1 Shown), in which 9 acquired deafness patients have been diagnosed, the peripheral blood samples of the deafness patients (experimental group, 9 people) and non-deaf individuals (9 people) in the family were extracted respectively.

[0038] The second step, sample exome sequencing and disease-caus...

Embodiment 2

[0043] Application of an autosomal dominant Dnajc17 gene mutant gene mutant in the preparation of a deafness diagnostic kit.

[0044] The nucleotide sequence of the autosomal dominant Dnajc17 mutant is shown in SEQ ID: NO: 2, and its specific PCR upstream and downstream primers are designed by Primer 5, and Invitrogen Company is responsible for primer synthesis. The purity is PAGE grade. The synthesized primers Use RNase free H2O to dissolve, the total concentration is 10 μM.

[0045] A diagnostic kit for an autosomal dominant Dnajc17 gene mutant comprising:

[0046] (a) DNA extraction system:

[0047] QIAmp Blood Kit;

[0048] (b) PCR system:

[0049] PrimeSTAR HS DNA Polymerase 50μL;

[0050] buffer 100μL;

[0051] dNTP Mixture (2.5mM each) 250μL;

[0052] Dnajc17 gene mutant specific amplification upstream primer (SEQ ID NO:3), 1 tube, 10 μM, 100 μL / tube;

[0053] Dnajc17 gene mutant specific amplification downstream primer (SEQ ID NO:4), 1 tube, 10 μM, 100 μL / tube; ...

Embodiment 3

[0056] Sequence Detection of Dnajc17 Gene in Peripheral Blood Cells of Deaf Patients

[0057] Genomic DNA samples from the peripheral blood of deaf patients were extracted using the QIAmp Blood Kit to ensure that the 260 / 280 ratio was above 1.8, and stored at -20°C.

[0058] The expression sequence of Dnajc17 was amplified by PCR, and the reaction system was as follows:

[0059] 5×PrimeSTAR Buffer (Mg 2+ Plus) 10 μl

[0060] dNTP mixture (2.5 mM each) 4 μl

[0061] Dnajc17 primer1 (10 μM) 1 μl

[0062] Dnajc17 primer2 (10 μM) 1 μl

[0063] gDNA template (100ng / μl) 1 μl

[0064] PrimeSTAR HS DNA Polymerase 0.5 μl

[0065] f 2 O 32.5 μl

[0066] PCR conditions: 95°C for 5min, (95°C for 30s; 55°C for 30s; 72°C for 2 min, 40 cycles), 72°C for 5min.

[0067] Sequence determination and result analysis:

[0068] Send the PCR reaction product of Dnajc17 to the biological company for sequence determination, and compare the obtained sequence with the reference sequence to find...

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Abstract

The invention discloses an autosomal dominant Dnajc17 gene mutant as well as application, a diagnostic kit and a diagnostic gene chip thereof. The mutation site of the autosomal dominant Dnajc17 genemutant is that the 401st nucleotide A of a Dnajc17 gene coding region on the human fifteenth chromosome is mutated into C (c.401A>C), so that the 134th amino acid of the protein sequence is mutated into alanine A (p.E134A) from glutamic acid E. Exome sequencing analysis and animal experiment verification are carried out on deafness family members; the Dnajc17 is found to have obvious correlation with the deafness occurrence, and is an important precipitating factor of hereditary hearing loss. The invention provides the diagnostic kit and the diagnostic gene chip for early diagnosis and early intervention treatment of deafness diseases, the diagnostic kit and the diagnostic gene chip have great clinical application value, and the diagnostic kit and the diagnostic gene chip can be used for screening high-risk groups with deafness diseases.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an autosomal dominant Dnajc17 gene mutant and its application, a diagnostic kit, and a diagnostic gene chip. Background technique [0002] Deafness is one of the most common diseases that cause human communication disorders, which brings great pain and inconvenience to patients. There are more than 400 million people in the world suffering from deafness, and about 40% of the elderly over the age of 65 suffer from deafness. Since the current treatment methods for deafness are still very limited, it is of great significance to detect and diagnose the occurrence of deafness diseases as early as possible. [0003] Genetic factors are an important cause of deafness. More than 400 kinds of hereditary syndromic deafness have been found, and more than 100 gene variations are related to the occurrence of deafness. The vast majority of deafness is sensorineural, which res...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156Y02A50/30
Inventor 刘东段旭初张鲁平
Owner NANTONG UNIVERSITY
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