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FMR1 (fragile x mental retardation 1) gene CGG repetition number and methylation detection kit and detection method

A detection kit and repeat number technology are applied in the field of FMR1 gene CGG repeat number and methylation detection kits, which can solve the problems of inaccurate detection results and achieve the effects of accurate interpretation, easy clinical application and promotion, and simple operation.

Active Publication Date: 2021-07-23
深圳会众生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to propose FMR1 gene CGG repeat number and methylation detection kit and detection method, aiming at solving the technical problem that the detection result of existing detection method is not accurate enough

Method used

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  • FMR1 (fragile x mental retardation 1) gene CGG repetition number and methylation detection kit and detection method
  • FMR1 (fragile x mental retardation 1) gene CGG repetition number and methylation detection kit and detection method
  • FMR1 (fragile x mental retardation 1) gene CGG repetition number and methylation detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Magnetic bead-labeled capture probes

[0061] The process for magnetic bead labeling of capture probes is as follows:

[0062] (1) Take two test tubes, add 20-100 μl (preferably 30 μl) of amino-modified magnetic beads (20g / L) solution respectively, and discard the supernatant;

[0063] (2) Add 100 μl of cross-linking agent (5%) to two test tubes, shake and mix once every 5-20 minutes (preferably 10 minutes) at room temperature, and shake and mix for 2-5 times (preferably 3 times) ), discard the supernatant;

[0064] (3) Add phosphate buffer solution (pH = 7-8, preferably pH = 7.4) to the two test tubes, shake and wash the magnetic beads, and discard the supernatant;

[0065] (4) Add F10 and R10 to the two test tubes, shake and mix once every 5-20 minutes (preferably 10 minutes), for a total of 2-8 times (preferably 5 times).

[0066] For the method of modifying the magnetic beads with amino groups, please refer to the prior art. The crosslinking agent may...

Embodiment 2

[0067] Embodiment 2. Acquisition of Target Fragment

[0068] The samples used in the detection kit and detection method of the present invention may be whole blood samples, dried blood spot samples, or oral epithelial cell samples. The above-mentioned samples can be extracted through corresponding extraction kits according to the instructions of the kits.

[0069] For the reaction system and reaction conditions of genomic DNA digestion, please refer to the prior art. Briefly, the digestion reaction process of genomic DNA is as follows:

[0070] (1) Take an appropriate amount of sample genomic DNA;

[0071] (2) Add PmlI, NaeI and Buffer to the reaction system according to the instructions;

[0072] (3) Carry out the enzyme digestion reaction at 37°C, and refer to the instruction manual for the reaction time;

[0073] (4) Store the digested product at 4°C for subsequent experiments.

Embodiment 3

[0074] Example 3. Capture Probes Capture Target Fragments

[0075] The process of capturing the target fragment by the capture probe includes pre-hybridization, blocking, hybridization and elution.

[0076] One), the process of pre-hybridization and sealing is as follows:

[0077] (1) Add 50-100 μl (preferably 60 μl) of prehybridization solution to the F10 capture probe and the R10 capture probe coupled to magnetic beads, and then add an equal volume of the blocking solution, and shake for 0.5 ~ 2 hours (preferably 1 hour);

[0078] (2) adding the phosphate buffer solution to wash;

[0079] (3) Add 100 μl of hybridization solution (Tris-HCl buffer, PH=7.4), and add the digested DNA sample, heat at 95°C for 5 minutes to decompose the DNA into single-stranded molecules, and shake the hybridization reaction at 40°C for 15~ 60 minutes (preferably 30 minutes);

[0080] (4) Discard the supernatant, add double distilled water to shake and rinse for 2 minutes, repeat three times; ...

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Abstract

The invention discloses an FMR1 (fragile x mental retardation 1) gene CGG repetition number and methylation detection kit and a detection method. The detection kit comprises a restriction enzyme, a magnetic bead and a capture probe. The restriction enzyme is used for enzyme digestion of genome DNA of a sample, the magnetic bead is connected with a first modification group, the capture probe is connected with a second modification group, and the second modification group is used for connecting the first modification group so as to couple the magnetic bead and the capture probe. The capture probe coupled with the magnetic bead is used for specifically binding to a DNA fragment subjected to enzyme digestion. The detection kit and the detection method are simple and convenient to operate, accurate in interpretation and easy to clinically apply and popularize.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a FMR1 gene CGG repeat number and methylation detection kit and detection method. Background technique [0002] The FMR1 gene is located on the X chromosome and is 38kb in length. The 5' untranslated region of the FMR1 gene contains CGG repeats. The repeat number of the CGG repeat sequence is polymorphic, and the repeat number may vary among different people. According to the number of CGG repeats, the researchers divided the FMR1 gene into four genotypes, namely wild type (CGG repeat number <45), intermediate type (CGG repeat number 45-54), pre-mutant type (CGG repeat number 55 ~200) and full mutants (CGG repeats >200). The CGG repeat number of normal people is below 45. Pre-mutation carriers (mostly males) have a certain possibility of suffering from Fragile X-associated tremor / ataxia syndrome (FXTAS), and women have a certain possibility of suffering from FXPO...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/683C12Q1/6869C12Q1/6883
CPCC12Q1/683C12Q1/6869C12Q1/6883C12Q2600/156C12Q2600/154C12Q2521/301C12Q2537/164C12Q2563/143C12Q2565/519C12Q2563/185
Inventor 张俊杰刘晶晶刘福平董玉莲
Owner 深圳会众生物技术有限公司
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