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A dna library for detecting and diagnosing pathogenic genes of ion channelopathies and its application

A technology of ion channelopathy and DNA library, applied in the field of DNA library, can solve the problems of timeliness and low throughput of multi-sample detection in gene detection area, reduce cost and labor intensity, wide coverage, and improve timeliness Effect

Inactive Publication Date: 2018-03-16
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional Sanger sequencing-based gene detection method has the disadvantage of low throughput, and only one amplified region can be detected in one reaction, which cannot meet the requirements of a large genetic detection area and multi-sample detection timeliness

Method used

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  • A dna library for detecting and diagnosing pathogenic genes of ion channelopathies and its application
  • A dna library for detecting and diagnosing pathogenic genes of ion channelopathies and its application
  • A dna library for detecting and diagnosing pathogenic genes of ion channelopathies and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. Reagents used in the method:

[0057] Ion AmpliSeq TM Library Kit 2.0, Ion PGM TM Template OT2 200 Kit v3, Ion Sequencing 200 Kit v2, Ion Xpress Barcode Adapters 1-16Kit, Ion 318 TM ChipKit v2

[0058] 2. Specimen collection and preservation

[0059] (1) Specimen collection: The specimen is the peripheral blood of the patient. Blood is 5ml of venous blood taken routinely, treated with EDTA anticoagulant.

[0060] (2) Storage: It can be detected immediately, stored at 4°C for one week, and stored at -80°C for more than one week.

[0061] 3. Detection steps and result analysis:

[0062] (1) Extraction of genomic DNA of specimens: DNA extraction of specimens was carried out according to the operation instructions of the blood DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0063] (2) Super multiplex PCR amplification and library building of the target detection region: with the whole exons of 74 genes involved in the present invent...

Embodiment 2

[0099] In the present invention, the primer pool contains 15 ion channelopathy pathogenic genes newly discovered by the inventor, as shown in the following table:

[0100]

[0101]

[0102] The newly discovered 15 ion channelopathy pathogenic genes come from the research accumulation and family investigation of the inventors over the years. The inventor first searched through the KEGG signaling pathway and the homologous gene library among the known ion channelopathy pathogenic genes to find the homologous genes of the known pathogenic genes. Then, targeted high-throughput sequencing was performed on the candidate homologous disease-causing genes in the large-scale case sample bank of Chinese Han patients accumulated over the years, and bioinformatics analysis was performed to screen and find the disease-causing mutations. Cases with pathogenic mutations were screened for follow-up and further family analysis, and the entire family of the patient was sequenced to verify ...

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Abstract

The invention discloses a deoxyribonucleic acid (DNA) library for detecting disease-causing genes of ion channel diseases by using a targeted high-flux semiconductor sequencing technology and application thereof. In particular, a primer pond is designed according to 74 disease-causing genes of ion channel diseases, a sample genome DNA is subjected to super-multiple polymerase chain reaction (PCR) amplification, and amplification products are sequenced by using the high-flux semiconductor sequence technology in order to find disease-causing mutation and lay bases on the aspects of genetics and molecular biology for clinical diagnosis.. The DNA library has the characteristics of accuracy, rapidness, flexibility and low cost. Through 74 gene detection areas, various common fatal ion channel diseases such as long QT syndrome, short QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, familial atrial fibrillation, sick sinus syndrome and cardiac conduction block can be detected. The DNA library has important significance and a clinical value to diagnosis and identification of the ion channel diseases.

Description

technical field [0001] The invention relates to a DNA library for detecting and diagnosing ion channelopathy pathogenic genes through targeted high-throughput semiconductor sequencing technology and its application. Specifically, according to the causative gene of ion channel disease, super multiplex PCR primers that can cover the exons of the above genes and adjacent regions are designed, and the genomic DNA of the sample is subjected to super multiplex PCR amplification, and the amplified products are analyzed using high-throughput sequencing technology. Sequencing, looking for pathogenic mutations, clarifying the genetic etiology of channelopathies, and providing a theoretical basis for genetics and molecular biology for clinical diagnosis, belongs to the genetic testing technology in the clinical testing technology in the field of biomedicine. Background technique [0002] Ion channels are a special kind of hydrophilic protein on cell membranes and organelles. They are t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B40/08C12Q1/68
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2600/16C40B40/08C12Q2531/113C12Q2535/122C12Q2537/143C12Q2565/629
Inventor 汪道文汪道武李宗哲
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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