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A kit and gene chip for detecting common mutations of atp7b gene

A technology of gene chips and kits, which is applied in the field of kits and gene chips for detecting common mutations of ATP7B gene, can solve the problems of difficult operation, large difference, unfavorable clinical promotion and application, etc., so as to reduce experimental cost, simple experimental operation, time-saving effect

Active Publication Date: 2022-03-18
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, domestic gene chip research has not been widely used in the field of molecular diagnosis, and there is no research and development and report of a clinical mutation detection chip for the ATP7B gene of hepatolenticular degeneration
In 2008, there was a hepatolenticular degeneration ATP7B gene mutation detection chip for the Czechoslovakian population, but this chip is based on the microarray primer extension reaction technology (arrayed primer extension reaction, APEX), need to target each mutation site It is difficult to design specific extension primers, which is not conducive to clinical application
In addition, most of the mutations detected by the chip are hotspot mutations in the Czech population, which is quite different from the mutation spectrum of patients in my country, so it does not have the value of introduction and application in China

Method used

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  • A kit and gene chip for detecting common mutations of atp7b gene
  • A kit and gene chip for detecting common mutations of atp7b gene
  • A kit and gene chip for detecting common mutations of atp7b gene

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preparation example Construction

[0049] The present invention further provides the preparation method of the gene chip, which comprises the following steps: synthesizing the probe for the above-mentioned mutation detection, dissolving the probe into a 100 μmol / L solution with DEPC water, and mixing with Mix the spotting solution with a final concentration of 10 μmol / L, and put it into a 384-well plate in a spotting matrix arrangement, 10 μl per well; paste a fence on the surface of the chip (preferably a multi-sample chip fence, Chengdu Boao Jingxin Biotechnology Co., Ltd. Co., Ltd. products), start Boao Jingxin PersonalArrayer16 personal sampling instrument and supporting software, run the cleaning program, clean the sampling needle with deionized water, and perform position correction according to the software operation instructions, and follow the attached figure 2 Matrix spotting; the spotting step is pre-spotting 20 times, and then pointing probes on the detection chip, the horizontal spacing between poi...

Embodiment 1

[0093] Design and modification of primers and probes

[0094] Primers were designed according to the common mutation sites of ATP7B, and after repeated screening and verification, a set of specific primer pairs for amplifying the target fragment containing the mutation sites was obtained (see Table 1). The length of the fragments amplified by these primers is concentrated between 80-300bp, and the length is moderate, and multiple asymmetric PCR amplification reactions can be completed under the same reaction conditions (divided into three groups, respectively performing multiple asymmetric PCR). Clear bands can be observed in 3% agarose gel electrophoresis, which can meet the needs of downstream hybridization. The 5' end of the reverse primer was fluorescently labeled with Cy3, and the primers were dissolved in DEPC water as a 100 μmol / L storage solution, and stored at -20°C in the dark. The working concentration of the forward primer was 5 μmol / L, and the concentration of th...

Embodiment 2

[0097] Preparation of gene chip for detection

[0098] The aldehyde-based substrate used in this example is the product of Chengdu Boao Jingxin Biotechnology Co., Ltd. Take the probe storage solution, mix it with the spotting solution at a ratio of 1:9 before spotting, so that the final concentration of the working solution is 10 μmol / L, and add it into the 384-well plate according to the spotting matrix, 10 μl per well. Paste fences (preferably multi-sample chip fences, products of Chengdu Boao Jingxin Biotechnology Co., Ltd.) on the surface of the chip, and use Boao Jingxin PersonalArrayer16 personal spotting instrument to spot the gene chip. Run the cleaning program, clean the sampling needle with deionized water, and perform position correction according to the software operation instructions; the sampling step is to pre-spot 20 times, and then point the probe on the detection chip, the horizontal distance between points is 300 μm, and the vertical The spacing is 400 μm; ...

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Abstract

The invention discloses a kit and a gene chip for detecting common mutations of the ATP7B gene, and relates to the field of biological detection and identification. The kit includes probes for detecting 28 common mutation sites of the ATP7B gene, and multiple asymmetric PCR primer pairs for the common mutation sites; the gene chip includes the primers and probes as described above. The probes are immobilized on a solid support. The invention can simply and quickly detect 28 common mutation sites of the ATP7B gene in the Chinese population, and perform higher-throughput population mutation detection.

Description

technical field [0001] The invention relates to the field of biological detection and identification, more specifically, to a kit and a gene chip for detecting common mutations of the ATP7B gene. Background technique [0002] Hepatolenticular degeneration (HLD), also known as Wilson disease (WD), is an autosomal recessive disorder of copper metabolism caused by ATP7B gene mutations. ATP7B gene mutations cause massive deposition of copper ions It is an important pathogenic factor, so early diagnosis and copper removal treatment are of great significance to the prognosis of patients. At present, the detection of ATP7B gene mutation based on Sanger sequencing has become an important reference index for the molecular diagnosis of hepatolenticular degeneration. [0003] At present, most of the mutations in the ATP7B gene still use the classic method of next-generation sequencing. Although this method can provide accurate detection results, because the ATP7B gene is large and ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6837C40B40/06C12N15/11
CPCC12Q1/6883C12Q1/6837C40B40/06C12Q2600/156
Inventor 黄坚周冬虎贾思雨李潇瑾
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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