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Probe combination for detecting hereditary hearing loss and application thereof

A probe and gene detection technology, which is applied in the fields of biomedicine and genetic engineering, can solve the problems of false positive or false negative, and achieve the effect of improving accuracy and overcoming allelic tripping

Pending Publication Date: 2020-08-07
UNIMED BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the WGA method will produce a certain allelic dropout (ADO), and there will be a certain possibility of false positive or false negative by directly detecting the mutation of the pathogenic gene site

Method used

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  • Probe combination for detecting hereditary hearing loss and application thereof
  • Probe combination for detecting hereditary hearing loss and application thereof
  • Probe combination for detecting hereditary hearing loss and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Capture probe set of hereditary deafness-related genes:

[0032] The genes that can be captured by the capture probe set (liquid phase capture probe) include:

[0033] Group 1: Coding regions and splicing sites of hereditary deafness-related genes GJB2, GJB3 and SLC26A4:

[0034] Group 2: SNPs within 2MB regions upstream or downstream of 3 genes

[0035] SNP screening principles: selecting data from the Thousand Genomes Project

[0036] (http: / / www.ncbi.nlm.nih.gov / variation / tools / 1000genomes / ), the minimum allele frequencies of CHB (Northern Han Chinese) and CHS (Southern Han Chinese) within 2 Mb upstream and downstream of each gene above are greater than 0.1 high-frequency mutation sites, remove polynucleotides (polyN) and polymorphic sites with GC content > 70% in the upstream and downstream 50bp sequences of the site, and select unique to compare to the human genome hg19, obtained after screening SNP loci.

[0037] The first group and the second group ...

Embodiment 2

[0038] Embodiment 2 target gene detection method

[0039] 1. Library construction and sequencing

[0040] 1. Sample DNA Preparation

[0041] Samples can be DNA extracted from blood or saliva, or single cells or a small number of cells, DNA products obtained through single-cell whole genome amplification (WGA);

[0042] 2. Sample DNA Fragmentation

[0043] About 50ng of genomic DNA and / or single-cell WGA products were disrupted by ultrasound (covaris), resulting in DNA fragments with a main length of 200-250bp.

[0044] 3. Genomic library construction using KAPA reagents

[0045]The library can be constructed using the commercially available reagent KAPA Hyper Prep Kit (KAPA, KK8504). The main steps include: end repair of the fragmented DNA and adding A to the tail, linker ligation and purification (the linker contains the Index sequence, and each sample uses Linkers containing different index sequences are connected, and the linker sequence is a T overhang), and finally a ...

Embodiment 3

[0055] Example 3 Non-invasive prenatal diagnosis of deafness gene GJB2

[0056] 1. Sample collection: Collect a family with GJB2 gene-related hereditary deafness (the pregnant woman is heterozygous for GJB2 c.299delAT, the father of the fetus is heterozygous for GJB2 c.235delC, the proband is compound heterozygous for GJB2 c.299delAT and GJB2 c.235delC) Pregnant women collect 16ml of peripheral blood within 10-14 weeks of pregnancy to capture fetal cells in peripheral blood; the biological father, pregnant woman, and proband patient collect saliva for genomic DNA extraction.

[0057] 2. According to the method of Example 1, use the probe combination of the present invention to carry out library construction and on-machine sequencing, and perform data analysis.

[0058] 3. The results of data analysis are as follows:

[0059] (1) Data quality control:

[0060]

[0061] For genomic DNA, the coverage rate of the coding region of the detected gene region is 100%, and only a f...

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Abstract

The invention belongs to the field of biomedicine and gene engineering. Probe combination is disclosed by the invention and can be used for identifying exon sequences of pathogenic genes GJB2, GJB3 and SLC26A4 aiming at hereditary hearing loss or SNP sites in upstream or downstream 2Mb of the genes. The probe combination is suitable for crowd diagnosis and screening, and comprises non-invasive prenatal detection or embryo pre-implantation detection. The invention also discloses a corresponding detection method, a detection kit and an application thereof. According to the invention, hereditaryhearing loss detection can be carried out, detection of all exons and splicing areas of pathogenic genes is realized, the coverage area is more comprehensive, family linkage analysis can be realized by combining genes adjacent to SNP sites, and especially, the accuracy of single-gene disease detection based on single or few cells can be improved.

Description

technical field [0001] The invention belongs to the fields of biomedicine and genetic engineering, and provides a probe combination for detecting hereditary deafness and its application. Background technique [0002] Deafness is a common physiological defect in humans, with an incidence of about 2%, and deafness caused by genetic factors accounts for more than 60%. The number of patients with hereditary deafness is increasing year by year all over the world, and there is at least 1 deaf patient in every 1000 newborns. Hereditary deafness can be divided into chromosomes: X-linked, autosomal recessive, autosomal dominant, and mitochondrial. According to clinical symptoms, it can be divided into: syndromic and non-syndromic deafness. Studies at home and abroad have shown that non-syndromic hearing loss (NSHL) accounts for more than 70% of the deaf population in hereditary deafness. In my country, non-syndromic hearing loss-related gene defects are commonly found in GJB2, GJB3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6858C12Q1/6883
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2600/166C12Q2535/122C12Q2545/113
Inventor 吴涵陈伟坚陈究成刘虎张红莉
Owner UNIMED BIOTECH SHANGHAI CO LTD
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