PCR (polymerase chain reaction) detection method for CGG repeat number of FMR1 (fragile X mental retardation 1) gene 5' terminal noncoding region

A non-coding region and gene technology, applied in the field of PCR detection of the number of CGG repeats in the non-coding region of the 5' end of the FMR1 gene, can solve the problems of mental decline, irritability, personality changes, etc.

Inactive Publication Date: 2011-11-16
北京康为世纪生物科技有限公司
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  • Summary
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  • Application Information

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Problems solved by technology

3. Emotional instability, anxiety, irritability, personality changes
4. Short-term memory loss, mental decline
Therefore, there is currently no satisfactory method for prenatal screening of FXS

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0027] Example 1. The method of genomic DNA sodium nitrite treatment

[0028] Reagents and buffers required:

[0029] a) 5 micrograms of genomic DNA.

[0030] b) 1M NaOH solution

[0031] c) 3M sodium acetate solution (pH5.3)

[0032] d) absolute ethanol

[0033] e) Sodium nitrite buffer solution, its composition is sodium nitrite 500mM, sodium acetate 26mM, acetic acid 52mM, sodium chloride 150mM

[0034] experimental method:

[0035] Add an equal volume of 1M NaOH to human genomic DNA. Place in a 70°C water bath for 1 hour and immediately place on ice. Then add an equimolar amount of HCL to neutralize the reaction system. After adding 1 / 10 volume of 3M sodium acetate (pH5.5) to stabilize the pH, then add twice the volume of absolute ethanol. Centrifuge the DNA in a benchtop centrifuge at 12,000 rpm. Dissolve the DNA pellet in 500ul of freshly prepared sodium nitrite buffer. Place in a water bath at 50°C for 16 hours and then add twice the volume of absolute ethano...

example 2

[0036] Example 2. PCR reaction

[0037] Prepare the PCR reaction system according to Beijing Kangwei Century LAmp DNA Polymerase MasterMix instructions, including: 2X LAmp DNA Polymerase MasterMix, genomic DNA 100ng obtained from Example 1, MgCl 2 1.5 mM, upstream primer (SEQ ID NO: 1): TCAGGCGCTCAGCTCCGTTTCGGTTTCA and downstream primer (SEQ ID NO: 2): AAGCGCCATTGGAGCCCCGCACTTCC: 1uM each. Betaine 2M. The thermal cycle program of PCR reaction is: 98°C for 5min; 35 cycles of 98°C for 10s-64°C for 30s-68°C for 2min; 68°C for 4min.

example 3

[0038] Example 3. (CCT) 4 and (CCG) 4 degenerate primer PCR reaction

[0039] Prepare the PCR reaction system according to Beijing Kangwei Century LAmp DNA Polymerase MasterMix instructions, including: 2X LAmp DNA Polymerase MasterMix, genomic DNA 100ng obtained from Example 1, MgCl 2 1.5 mM, upstream primer TCAGGCGCTCAGCTCCGTTTCGGTTTCA (SEQ ID NO: 1), (CCT) 4 Primers CCTCCTCCTCCT (SEQ ID NO: 3) and (CCG) 4 Primers CCGCCGCCGCCG (SEQ ID NO: 4) were 1 uM each. Betaine 2M. The thermal cycle program of PCR reaction is: 98°C for 5min; 35 cycles of 98°C for 10s-64°C for 30s-68°C for 2min; 68°C for 4min.

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Abstract

The invention relates to an auxiliary laboratory diagnosis method for some hereditary diseases caused by X chromosome FMR1 (fragile X mental retardation 1) gene abnormity. The method is established aiming to improve the accuracy of clinical diagnosis by using a molecular biological detection method as an auxiliary means for clinical examination, and to provide a new detection means for prenatal screening for related hereditary diseases.

Description

[0001] Field of the invention: [0002] The invention relates to a laboratory auxiliary diagnosis method for several genetic diseases caused by X chromosome FMR1 gene abnormality. The establishment of this method aims to use molecular biology detection methods as an auxiliary means of clinical examination, improve the correct rate of clinical diagnosis, and provide a new detection method for prenatal screening of related genetic diseases. Background of the invention: [0003] Fragile X syndrome, Fragile X-related ataxia syndrome, and Fragile X-related ovarian insufficiency [0004] Fragile X syndrome (fragile x syndrome, FXS), also known as fragile X syndrome or Martin-Bell syndrome, was first reported in the late 1960s and is an X-linked incomplete dominant genetic disease. The patient began to have symptoms of abnormal mental development and a series of abnormal appearance signs from childhood or adolescence. The main symptoms are 1. Mental retardation, poor calculation ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 任军任黎明王春香高建恩
Owner 北京康为世纪生物科技有限公司
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