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Method for detecting FUS gene mutation and TARDBP gene mutation

A technology for genome and sequencing results, applied in the field of molecular biology and biology, can solve the problems of time-consuming and laborious operation, high use cost, expensive equipment, etc., and achieve the effect of low cost, high accuracy and specificity, and simple operation

Pending Publication Date: 2019-12-06
SHENZHEN CITY BAOAN DISTRICT MATERNAL & CHILD HEALTH HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the above methods are high cost of use, expensive instruments, time-consuming and labor-intensive or complicated operations

Method used

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  • Method for detecting FUS gene mutation and TARDBP gene mutation
  • Method for detecting FUS gene mutation and TARDBP gene mutation
  • Method for detecting FUS gene mutation and TARDBP gene mutation

Examples

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Effect test

Embodiment 1

[0077] Example 1: Mutation detection experiment of mutation hot spot region of ALS pathogenic gene FUS (NM_004960.3)

[0078] Method establishment

[0079] Obtain the mutation hotspot region of the ALS pathogenic gene FUS (NM_004960.3), including all 10 ALS pathogenic mutations on ClinVar (G206S; R216C; R495X; G507D; H517Q; R518K; R521C; R521G; R521H; R524W) and Possible de novo mutations. PCR-HRM primers were designed to cover the mutation hotspots mentioned above. The primer sequences and the lengths of amplified fragments are shown in Table 1 (SEQ ID 1-6).

[0080] Detection of FUS gene mutation

[0081] S1. Using general methods or kits to extract genomic DNA from peripheral blood leukocytes;

[0082] S2, using the kit, using genomic DNA as a template to perform PCR-HRM amplification reaction;

[0083] The amplification reaction system adopts Eco Real-Time PCR System (Illumina), and the reaction system is 10 μL, including: template DNA (20ng), 1×PCR buffer, dNTP (0.2mM...

Embodiment 2

[0107] Example 2: Mutation detection experiment in mutation hotspot region of ALS pathogenic gene TARDBP (NM_007375.3)

[0108] Method establishment

[0109] Obtain the mutation hotspot region of the ALS pathogenic gene TARDBP (NM_007375.3), including all 16 ALS pathogenic mutations on ClinVar (D169G; c.*83T>C; c.*697G>A; G290A; G294V; G294A; G295S; G298S; A315T; Q331K; M337V; Q343R; G348C; A382T; G384R; W385G) and possible de novo mutations. PCR-HRM primers were designed to cover mutation hot spots. The primer sequences and the length of the amplified fragments are shown in Table 1 (SEQ ID 7-18).

[0110] Detection of TARDBP gene mutation

[0111] S1. Using general methods or kits to extract genomic DNA from peripheral blood leukocytes;

[0112] S2, using the kit, using genomic DNA as a template to perform PCR-HRM amplification reaction;

[0113] The amplification reaction system adopts Eco Real-Time PCR System (Illumina), and the reaction system is 10 μL, including: temp...

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Abstract

The invention discloses a method for detecting FUS gene mutation and TARDBP gene mutation. The invention establishes an analysis method based on PCR-HRM (high-resolution melting). The method can rapidly identify all known mutation and novel mutation in an amplification area and provides important technical means for discovery, genetic counseling and mechanism research of pathogenic genes of rare diseases such as ALS. Besides, the method is high in detection efficiency, high in accuracy and specificity, low in cost and simple to operate.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a method for detecting FUS gene mutation and TARDBP gene mutation. Background technique [0002] Amyotrophic lateral sclerosis (ALS) is a progressive disease characterized by the degeneration of motor neurons in the cortex, brainstem, and anterior spinal cord. Clinical manifestations of patients usually have upper motor neuron paralysis and lower motor neuron paralysis. ALS typically first affects neurons in one area (head / neck / thorax / lumbar) and then gradually spreads to other areas. Typical presentations include limb and bulbar onset, and less often respiratory failure. Usually the clinical presentation progresses gradually over time without periods of remission. Eventually the patient gradually becomes disabled and dies. There is currently no cure. Treatment is mainly supportive care and palliative care. [0003] The aim is to improve the quality of li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6869C12Q1/6827
CPCC12Q1/6883C12Q1/6869C12Q1/6827C12Q2600/156C12Q2531/113C12Q2549/119C12Q2527/107
Inventor 王丰许雪青符胜煜李发科谢小红雷佳凡郭波陈献
Owner SHENZHEN CITY BAOAN DISTRICT MATERNAL & CHILD HEALTH HOSPITAL
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