Kit for detecting related gene FMR1 of fragile X chromosome syndrome (FRAX) and application of kit

A technology for detection kits and syndromes, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of long time, large demand for samples, complicated technical operations, etc., and achieves ingenious design and rapid detection. Effect

Active Publication Date: 2015-03-25
SHANGHAI CHROMYSKY MEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current cytogenetic detection, RFLP linkage analysis, and DNA hybridization analysis methods take a long time, require a large number of samples, and are complicated in technical operations, making it difficult to widely carry out
However, based on PCR amplification, capillary electrophoresis and sequencing analysis, due to the difficulty of amplification of (CGG)n, the success rate is not high, so the development is limited.

Method used

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  • Kit for detecting related gene FMR1 of fragile X chromosome syndrome (FRAX) and application of kit
  • Kit for detecting related gene FMR1 of fragile X chromosome syndrome (FRAX) and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: DNA preparation

[0047] (1) Preparation of HEK293 genomic DNA for testing

[0048] Routine culture collection 10 6 Genomic DNA was extracted from HEK293 cells (ATCC) with a Qiagen genome mini-extraction kit, and the concentration was adjusted to 20 ng / μL as a normal reference.

Embodiment 2

[0049] Example 2: Design and synthesis of FRA(X) mutant gene amplification primers and competitive primers

[0050] Synthetic primers were designed according to FMR1 (CGG)n and surrounding areas. The gene-specific primers on the far side of the FMR1 (CGG)n 5' flank (covering the HpaII methylation-sensitive sequence) are SEQ ID NO: 1; FMR1 (CGG)n 3' flank The gene-specific primers on the distal side (covering HpaII methylation-sensitive sequences) are SEQ ID NO: 2, and labeled with FAM fluorescent dye; The neutral primer is SEQ ID NO:3, and is labeled with HEX fluorescent dye. The amplification primer sequences located within the (CGG)n sequence are SEQ ID NO: 4 (marked HEX) and SEQ ID NO: 5 (marked ROX), while introducing SEQ ID NO: 6 as a competitive primer to improve specificity ( Table 1).

[0051] Table 1: FRA(X) Mutant Gene Amplification Primers and Competitive Primers

[0052] .

[0053] In the present invention, HpaII enzyme digestion and control pretreatme...

Embodiment 3

[0055] Example 3: FMR1 gene mutation detection

[0056] (1) DNA HpalI digestion reaction;

[0057] Add 100 ng of DNA template, 1 μL of Hpa1I (NEB) buffer, 0.2 μL of HpaII to tube A, add water to 10 μL, 37°C 2Hr; 80°C, 20 min.

[0058] Add 100 ng of DNA template to tube B, 1 μL of HpaII (NEB) buffer, add water to 10 μL, and incubate at 37°C for 2Hr; 80°C for 20 min.

[0059] (2) FMR1 gene mutation amplification

[0060] Tube 1 reaction system: Tube A treatment template 1 μL, KOD-FX enzyme 1 unit, 2× KOD-FX buffer 5 μL, 2.5 mM dNTPs 1 μL, first tube primer mix 1 μL, add water to 10 μL.

[0061] Tube 2 reaction system: tube B treatment template 1 μL, KOD-FX enzyme 1 unit, 2× KOD-FX buffer 5 μL, 2.5 mM dNTPs 1 μL, second tube primer mix 1 μL, add water to 10 μL.

[0062] Tube 3 reaction system: Tube A treatment template 1 μL, KOD-FX enzyme 1 unit, 2× KOD-FX buffer 5 μL, 2.5 mM dNTPs 1 μL, tube 3 primer mix 1 μL, add water to 10 μL.

[0063] Tube 4 reaction system: tube B t...

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Abstract

The invention belongs to the technical field of detection of disease-related gene mutation, and in particular discloses a kit for detecting related gene FMR1 mutation of fragile X chromosome syndrome (FRAX). The kit consists of FRAX related gene FMR1 mutation detection probe combinations: namely polymerase chain reaction (PCR) primer groups SEQ ID NO. 1 and 2, SEQ ID NO. 1 and 3, SEQ ID NO. 1, 4 and 6 as well as SEQ ID NO. 1, 5 and 6. The detection steps are the follows: extracting to-be-detected genome DNA; displaying a possible methylation difference by virtue of HpaII enzyme digestion and control preprocessing; carrying out PCR amplification on the preprocessed genome DNA by virtue of a four-color fluorescence PCR primer; carrying out capillary electrophoresis; and according to a capillary electrophoresis curve, analyzing the FMR1 (CGG) n length and methylation situation of FRAX related gene in the genome DNA. The kit disclosed by the invention is fast, accurate, simple and convenient to use, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of disease-related gene mutation detection, and in particular relates to a FRA X syndrome-related gene FMR1 mutation detection kit and application thereof. Background technique [0002] Fragile X (Fra X) syndrome causes intellectual disability in patients (Martin-Bell syndrome). The incidence of this disease in males is 1 / 1000~1 / 1500, second only to congenital stupidity. Fragile X syndrome is caused by mutations in the body's X chromosome formation process. Mainly manifested as moderate to severe mental retardation, other common features include children with height and weight above normal, rapid growth, prominent forehead, hypoplasia in the middle of the face, large and protruding mandible, large ears, high arched palate, and thick lips , protruding lower lip, another important manifestation is large orchidism. Some patients also have ADHD, aggressive behavior or autism, moderate to severe mental retarda...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2545/113C12Q2563/107C12Q2565/125
Inventor 赵书民赵翊均陈金中周巍
Owner SHANGHAI CHROMYSKY MEDICAL RES
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