Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof

A technology for optic neuropathy and mutation sites, which is applied in the field of gene chip detection to achieve the effects of good clinical promotion value, shortened detection time, and shortened diagnosis time.

Inactive Publication Date: 2009-08-05
ANHUI MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Due to the large number of LHON-related mtDNA mutation sites, conventional genetic diagnosis methods have been difficult to meet the requirements. Theref...

Method used

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  • Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof
  • Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof
  • Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Probe Design and Chip Preparation

[0046] (1) Design of probes on the chip:

[0047] According to the 32 LHON-related mtDNA mutation sites and 11719G>A polymorphism sites reported in the MITOMAP database, and referring to the revised Cambridge mtDNA reference sequence (revised Cambridge Reference Sequence, rCRS), 33 pairs of oligonucleotide probes were designed and screened. Needle, so that the Tm value of different probes is located at (45±5) °C as much as possible, the length of the probe is located at 14-20bp, so that the gene locus detected by each pair of probes is located in the middle or near the middle of the probe sequence, so that the probe The binding site to the corresponding single-stranded target gene should be as close as possible between the 5' end and the middle region of the single-stranded target gene to ensure hybridization efficiency. The 5' end of the probe has an amino modification group, so as to combine with the aldehyde group modifi...

Embodiment 2

[0055] Example 2 Mutation detection of samples

[0056] (1) Preparation, labeling and processing of samples:

[0057] DNA extraction kits from Qiagen Company were used to extract DNA from samples from patients with Leber's hereditary optic neuropathy or suspected patients, and set aside.

[0058] PCR amplification and labeling treatment of samples: Under the same thermal cycle, through three sets of multiplex PCR (the nucleotide sequences of ID NO.66-71, ID NO.72-79, ID NO.80-87 were As three sets of multiplex PCR primers) to amplify 11 target genes, these 11 target genes cover 32 mtDNA mutation sites and 11719G>A polymorphism sites related to Leber's hereditary optic neuropathy. In 25ul PCR reaction system (amplification system includes 2.5ul 10*PCR buffer, each 200uM of dATP, dGTP, dCTP, dTTP, 1.5mM MgCl2 , 1.5U DNA TagE (Takara), and primers ranging from 0.05-0.5uM, add 100ng sample DNA to each reaction, and prepare 11 fluorescently labeled sample DNA target fragments thro...

Embodiment 3

[0072] The assembly of embodiment 3 kits

[0073] The gene chip was prepared according to Example 1, and the PCR primers of SEQ ID NO.66-87 recorded in Example 2 were subpackaged, assembled with the prepared gene chip, hybridization solution, and nucleic acid hybridization washing solution, and then packaged to obtain a kit.

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Abstract

The invention relates to a gene chip, and discloses an integrated detection gene chip of an mtDNA mutation site related to Leber genetic optic neuropathy, as well as the preparation and the application thereof. A specific oligonucleotide detecting probe of the mtDNA mutational site related to the Leber genetic optic neuropathy and a specific oligonucleotide detecting probe of a mononucleotide polymorphyism site mtDNA 11719G>A are fixed on the carrier surface lattice of the gene chip. The gene chip has the advantages of time saving, low cost, simple operation and the like, has 32 mutation sites related to the Leber genetic optic neuropathy in a specific detecting clinical sample only through one-time PCR amplification and one-time crossing reaction, and is suitable for the gene diagnosis of the Leber genetic optic neuropathy.

Description

technical field [0001] The invention relates to a gene chip, in particular to a detection gene chip related to Leber's hereditary optic neuropathy and its preparation and application. Background technique [0002] Leber's hereditary optic neuropathy (LHON) is the most common hereditary optic atrophy disease, characterized by bilateral acute or subacute central vision loss, mainly affecting adolescent males. In 1871, Theodor Leber first reported a LHON family and described the clinical features of LHON in detail. In the following period, it was believed that the genetic pattern of LHON was associated with the X chromosome (X-linked), until Erickson proposed in 1972 that the inheritance of LHON was more inclined to maternal inheritance, and he believed that there might be a potential mitochondrial DNA (mtDNA) ) mutations are associated with LHON. In 1988, Wallace and his colleagues reported for the first time that LHON was associated with the 11778G>A mutation of the MTND...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 杜卫东赵建龙张学军曹慧敏金庆辉陈刚
Owner ANHUI MEDICAL UNIV
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