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A method for high-throughput detection of Parkinson's disease-causing gene mutations

A technology for Parkinson's disease and pathogenic genes, applied in the field of high-throughput detection of Parkinson's disease pathogenic gene mutations, can solve the problems of cost and time-consuming that cannot meet large-scale sequencing, large amount of DNA, long experimental period, etc. Comprehensive coverage, high accuracy and highly targeted effects

Active Publication Date: 2020-12-18
上海昂朴生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, molecular diagnosis in the past relied on Sanger sequencing. In conventional generation sequencing, multiple PCRs are required for each gene. The amount of DNA used is large, the cost is high, and the experiment cycle is long. It is often necessary to detect multiple candidate genes one by one, which is costly and time-consuming. Cannot meet the needs of large-scale sequencing

Method used

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  • A method for high-throughput detection of Parkinson's disease-causing gene mutations
  • A method for high-throughput detection of Parkinson's disease-causing gene mutations
  • A method for high-throughput detection of Parkinson's disease-causing gene mutations

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Embodiment 1

[0079] In this embodiment, Miseq sequencing technology is used to detect the genomic DNA in the plasma of the subject. The specific steps are as follows:

[0080] 1. According to the instructions, use Roche's High Pure PCR Template Preparation Kit to extract the genomic DNA in the plasma of the subject.

[0081] 2. Use an ultrasonic breaker to break the genomic DNA into small fragments of about 500bp.

[0082] In this example, 3 μg of DNA was fragmented using a Covaris sonicator according to standard operations.

[0083] 3. Use Agencourt AMPure XP magnetic beads to purify the sample, the volume ratio of magnetic beads to sample is 1.5:1, and elute with 50 μl nuclease-free water. The specific operation is as follows:

[0084]

[0085]

[0086] 4. Use T4 DNA polymerase, Klenow DNA polymerase, T4PNK for end repair. The reaction system is as follows:

[0087] ingredients Volume (μl) DNA samples 48 Nuclease-Free water 35.2 10×End Repair Buffer ...

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Abstract

The invention relates to a high-throughput detection method of Parkinson's disease causing gene mutations (PDCap), which includes the steps of extracting genome DNA of a detection object; quantifying the extracted genome DNA, and collecting 3 Mu g to establish a library; quantifying the library; performing online sequencing; analyzing data to obtain related information of disease causing sites. Compared with the prior art, the method of the invention enables mutation conditions of multiple Parkinson's disease related causing mutation sites during single sequencing, and has the advantages of high sensitivity, good specificity, good coverage, high throughput, high accuracy and the like.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a high-throughput method for detecting Parkinson's disease-causing gene mutation (PDCap). Background technique [0002] Parkinson's disease (PD), also known as paralysis agitans, is mainly caused by the reduction of dopaminergic neurons in the substantia nigra of the midbrain. It is a common degenerative disease of the central nervous system in middle-aged and elderly people. The most common extrapyramidal disease. The prevalence of Parkinson's disease in the whole population is about 0.3%. As a typical chronic disease of the elderly, the prevalence of Parkinson's disease increases exponentially in the elderly population, and the prevalence rate of the elderly population over 65 years old is 1% to 2%. , 3% to 5% over the age of 85, and there is a trend that men are slightly more than women. The main clinical features of the disease are resting tremor, slowness and decrease ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2535/122
Inventor 王坚王曦路孙一忞陈静邬剑军丁正同
Owner 上海昂朴生物科技有限公司