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Constructing method of genetic testing library for inherited arrhythmias and kit for inherited arrhythmias

A gene detection and arrhythmia technology, which is applied in the field of gene detection library construction methods and kits, can solve the problems of the biased influence of subsequent sequencing data and uneven amplification of target regions.

Inactive Publication Date: 2019-04-09
ANNGEEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the multiplex PCR method is used to amplify the target region during the sample library preparation process, there will be amplification bias, resulting in uneven amplification of the target region, which will have a biased impact on subsequent sequencing data

Method used

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  • Constructing method of genetic testing library for inherited arrhythmias and kit for inherited arrhythmias
  • Constructing method of genetic testing library for inherited arrhythmias and kit for inherited arrhythmias
  • Constructing method of genetic testing library for inherited arrhythmias and kit for inherited arrhythmias

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Experimental program
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Effect test

Embodiment 1

[0098] Four peripheral blood samples were used for library construction, and the entire coding region and variable splicing region (20 bp extension from exon to intron) of KCNH2, KCNQ1, SCN5A, RYR2, CASQ2, TRDN, CALM1 genes were used as targets The primer pool in the region was subjected to multiple reaction PCR, combined with the high-throughput sequencing platform Miseq for DNA sequencing, and then detected point mutations (SNP) and small fragment insertion-deletion (InDel). The specific operation process is as follows:

[0099] (1) Nucleic acid extraction and quality inspection: After nucleic acid extraction and quality inspection of peripheral blood samples, it is required to meet certain quality control standards: DNA integrity is good, no protein RNA pollution, DNA concentration ≥ 20ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230>2.0; total DNA input amount: 0.1μg-1μg.

[0100] (2) Library preparation: Using the above DNA, make up to 52.5 μl with TRIS-EDTA after sampli...

Embodiment 2

[0109] 16 buccal swab samples were used for library construction, using the full coding region and variable splicing region (20 bp extension from exon to intron) of KCNH2, KCNQ1, SCN5A, RYR2, CASQ2, TRDN, CALM1 genes as targets The primer pool in the region was subjected to multiple reaction PCR, combined with the high-throughput sequencing platform Nextseq550AR for DNA sequencing, and then detected point mutations (SNPs) and small fragment insertion deletions (InDels). The specific operation process is as follows:

[0110] (1) Nucleic acid extraction and quality inspection: After nucleic acid extraction and quality inspection of peripheral blood samples, it is required to meet certain quality control standards: DNA integrity is good, no protein RNA pollution, DNA concentration ≥ 20ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230>2.0; total DNA input amount: 0.1μg-1μg.

[0111] (2) Library preparation: Using the above DNA, make up to 52.5 μl with TRIS-EDTA after sampling, per...

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Abstract

The invention discloses a constructing method of a genetic testing library for inherited arrhythmias and a kit for inherited arrhythmias, and relates to mutation of KCNH2, KCNQ1, SCN5A, RYR2, CASQ2, TRDN and CALM1 genes. Target regions are exon regions of coded amino acid of the seven genes, 20 base regions on the upstream of an exon and 20 base regions on the downstream of the exon; for ensuringall coverage of the target regions, after a preparation library is adopted, a target region library is obtained by a hybridization probe capturing method; then the library is amplified by adopting anLMPCR method; the library is purified to obtain a sample library for sequencing. The constructing method comprises simple and quick steps, so that the constructing cost of the library is effectively reduced; in addition, related genes of arrhythmia are covered; by combination with an illumine high throughput sequencer, the mutation of the related genes can be quickly and accurately obtained; the constructing method and the kit have important significance for the inherited arrhythmias.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and kit for constructing a gene detection library for hereditary arrhythmia. Background technique [0002] Inherited arrhythmia (Inherited Arrhythmia, DCM) is a group of genetic diseases with the risk of syncope or sudden death caused by potentially malignant arrhythmia, most of which are caused by gene mutations of ion channels involved in the regulation of cardiac action potential. The main disease classifications include Long QT syndrome (Long QT Syndrome, LQTS), Brugada syndrome (Brugada Syndrome, BrS), catecholamine-sensitive ventricular tachycardia (Catecholaminergic Polymorphic Ventricular Tachycardia, CPVT), short QT syndrome (Short QT Syndrome , SQTS), idiopathic ventricular fibrillation (Idiopathic Ventricular Fibrillation, IVF) and progressive cardiac conduction system disease (Progressive Cardiac Conduction Disease, PCCD). Most of the patients with th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6883C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6883C12Q2600/106C12Q2600/156C40B50/06C12Q2525/191C12Q2531/113C12Q2537/159
Inventor 扶媛媛周洋曹彦东马懿王苏
Owner ANNGEEN BIOTECHNOLOGY CO LTD
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