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Gene detection library construction method and kit for hereditary dilated cardiomyopathy

A technology for dilated cardiomyopathy and gene detection, which is applied in chemical libraries, biochemical equipment and methods, and microbial determination/inspection.

Pending Publication Date: 2019-04-05
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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  • Gene detection library construction method and kit for hereditary dilated cardiomyopathy
  • Gene detection library construction method and kit for hereditary dilated cardiomyopathy
  • Gene detection library construction method and kit for hereditary dilated cardiomyopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Four peripheral blood samples were used for library construction, using the full coding region and The variable splicing region (20bp extension from exon to intron) is used as the primer pool of the target region for multiple reaction PCR, combined with the high-throughput sequencing platform Miseq for DNA sequencing, and then detecting point mutations (SNP) and small fragment insertions Missing (InDel). The specific operation process is as follows:

[0122] (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.

[0123] (2) Library preparation: Using the above DNA, make up to 52.5 μl with TRIS-EDTA after sampling, perform ultrasonic fragmentation on the covaris instrument acc...

Embodiment 2

[0132]16 buccal swab samples were used for library construction, using the full coding region and The variable splicing region (20bp extension from exon to intron) is used as the primer pool of the target region for multiple reaction PCR, combined with the high-throughput sequencing platform Nextseq550AR for DNA sequencing, and then detecting point mutations (SNP) and small fragment insertions Missing (InDel). The specific operation process is as follows:

[0133] (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.

[0134] (2) Library preparation: Using the above DNA, make up to 52.5 μl with TRIS-EDTA after sampling, perform ultrasonic fragmentation on the covaris instrument acco...

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Abstract

The invention discloses a gene detection library construction method and a kit for hereditary dilated cardiomyopathy, and relates to mutation of ANKRD1, BAG3, LDB3, LMNA, MYBPC3, MYH6, MYH7, RBM20, SCN5A, TCAP, TNNC1, TNNI3, TNNT2, TPM1 , TTN and VCL genes. Target regions include exon regions of coded amino acids of the 16 genes and 20 basic group regions of each of the upstream and downstream ofeach exon; in order to ensure the full coverage of the target regions, a target region liberty is acquired according to a hybridization probe capture method after library preparation and then amplified by the LMPCR (ligation mediated polymerase chain reaction) method, and a sample library for sequencing is acquired after library purification. The library construction method has the advantages thatthe steps are simple and fast, the library construction cost is reduced effectively, arrhythmia related genes are included, related gene mutations can be acquired quickly and accurately through an Illumina high-throughput sequencer, and accordingly, the library construction method is important for the hereditary dilated cardiomyopathy.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and a kit for constructing a gene detection library of hereditary dilated cardiomyopathy. Background technique [0002] Hereditary dilated cardiomyopathy (Dilated Cardiomyopathy, DCM) is a cardiomyopathy with multiple causes, characterized by myocardial dilation and systolic dysfunction, and eventually leads to heart failure. The incidence rate is about 1 / 250, and about 20%-35% of DCM patients are familial, and most of the rest are sporadic DCM. The penetrance of hereditary DCM is positively correlated with age, and within the same family, there may be large differences in the age of onset and clinical manifestations of patients. DCM may also be accompanied by muscular dystrophy, conduction system disease, and potentially fatal cardiac arrhythmias. [0003] The main clinical symptoms of DCM include arrhythmia, dyspnea, fatigue, fainting, and sweating of legs and...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6858C12Q1/6883
CPCC12Q1/6858C12Q1/6883C40B50/06C12Q2600/156C12Q2531/113C12Q2535/122
Inventor 周洋扶媛媛曹彦东马懿王苏
Owner ANNGEEN BIOTECHNOLOGY CO LTD
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