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OSBPL2 mutant gene as well as identification method and detection kit thereof

A detection kit and identification method technology, which is applied in the field of hereditary non-syndromic deafness-related genes, its identification method and detection kit, can solve the problems of time-consuming and laborious, and achieve the effect of convenient method and cost reduction

Inactive Publication Date: 2014-04-30
NANJING MEDICAL UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the traditional method of mapping genes for monogenic hereditary diseases is used, it will be a time-consuming and laborious process because the linkage region will involve known genes, unknown genes and predicted genes

Method used

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  • OSBPL2 mutant gene as well as identification method and detection kit thereof
  • OSBPL2 mutant gene as well as identification method and detection kit thereof
  • OSBPL2 mutant gene as well as identification method and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Taking NSHL as an example, the inventor detected a new deafness-related gene (flow chart as shown in figure 1 shown), which includes the following steps:

[0057] 1) The inventor collected a 7-generation autosomal dominant NSHL pedigree, and the results of consultation and hearing tests showed that all patients in the pedigree showed late-onset, progressive moderate and severe non-syndromic deafness. The peripheral blood of the family members was collected and genomic DNA was extracted.

[0058] 2) Using human genome exon capture technology to perform exon capture and analysis on the whole genome DNA of 3 patients and 1 normal control in the family described in 1), and obtain the mutation sites of candidate hereditary deafness-related genes.

[0059] 3) Combining conventional sequencing methods to identify the mutation sites of candidate genetic deafness-related genes: a. Perform genetic co-segregation verification on the rest of the samples in the family that did not ...

Embodiment 2

[0061] Example 2: Sequencing the causative genes of this NSHL family using human genome exome capture

[0062] Subsequently, the inventors used Agilent SureSelect Human All Exon Kit (38M) combined with Solexa high-throughput sequencing technology to sequence the exome sequences of 3 patients and 1 normal person in the NSHL family described in Example 1, and successfully A new NSHL-associated gene, a mutant of the OSBPL2 gene, was discovered. The specific operation steps are as follows:

[0063] 1) Randomly break the genomic DNA into fragments of about 150-200bp, and then connect adapters to both ends of the fragments to prepare a hybrid library (see the Illumina / Solexa standard library construction instructions provided by http: / / www.illumina.com / ; Accurate whole human genome sequencing using reversible terminator chemistry. Nature 2008, 456:53-59).

[0064] 2) After purification, the library was subjected to linear amplification of Ligation-mediated PCR (LM-PCR) and hybridi...

Embodiment 3

[0094] Example 3: Further verification of the c.153del CT mutation site of the OSBPL2 gene

[0095] The inventor verified the c.153del CT mutation site of the OSBPL2 gene by combining conventional sequencing methods: ① Verify the genetic co-segregation of all samples in the family described in Example 1; ② Verify the exon and exon of the ① verified gene PCR amplification and detection of the sequence of the sub-intron junction region; ③ Expand the sample size (normal controls outside the family, small autosomal dominant families, sporadic deaf cases) verification.

[0096] 1) Amplify the mutation by PCR

[0097] Genomic DNA was prepared from the peripheral blood samples of all patients in the family according to the method in Example 1. After the concentration was adjusted to 25ng / μl, PCR reaction was carried out.

[0098] PCR primers were designed by Primer v5.0, and the sequences are as follows:

[0099] Upstream primer: CTGGATCTCACTCACATTCT (SEQ ID NO: 21)

[0100] Down...

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Abstract

The invention discloses an OSBPL2 mutant gene as well as an identification method and a detection kit thereof. The OSBPL2 mutant gene has the deletion of nucleotide C and T between 152-bit nucleotide C and 155-bit nucleotide C in an exon 3 region of a human OSBPL2 gene sequence, and / or 583-bit nucleotide C in an exon 7 region of the human OSBPL2 gene sequence, is mutated to nucleotide A. By adopting the OSBPL2 mutant gene as well as the identification method and the detection kit thereof, a pathogenic gene of a hereditary disease is discovered by utilizing a human genome exon trapping technology; the identification method is simple and convenient; in addition, as only one or a small quantity of gene mutation sample is needed to trap a human genome exon sequence, the cost is greatly lowered; gene identification has an important value to genetic diagnosis of NSHL (Non-Syndromic Hearing Loss) susceptible and occurrence populations and has an important significance of exploring NSHL pathogenesis and opening up a novel therapy approach.

Description

technical field [0001] The invention belongs to genetics and molecular biology, and specifically relates to an identification method of a genetic disease-causing gene, in particular to a genetic non-syndromic deafness-related gene, an identification method and a detection kit. Background technique [0002] Deafness is a relatively common human sensory system defect and a common disease that affects human health and causes disability. The etiology of deafness is complex, and genetic factors play an important role. More than 60% of congenital deafness is caused by genetic factors [1-2] . Studies have shown that more than 60 different types of gene variations distributed in the nucleus and mitochondria are involved in the occurrence of hereditary non-syndromic hearing loss (NSHL). These genes are mainly arranged on autosomes, and only a few Located in sex chromosomes and mitochondrial DNA, they present a typical single-gene inheritance and mitochondrial maternal inheritance i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C12Q1/68
Inventor 曹新邢光前魏钦俊姚俊陈智斌鲁雅洁
Owner NANJING MEDICAL UNIV
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