Primer and method for detecting FGD1 gene mutation

A sequencing primer, FGD1-EXON-3-F technology, applied in the field of life sciences and biology, can solve problems such as interference signal transmission system, spatial structure change, etc., and achieve the effect of difficult detection, low cost and high cost

Inactive Publication Date: 2019-04-16
杭州艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in the FGD1 gene can lead to changes in the spatial structure of the encoded GEF domain, thereby interfering with the normal signal transmission system of FGD1 / Cdc42, leading to the occurrence of AAS

Method used

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  • Primer and method for detecting FGD1 gene mutation
  • Primer and method for detecting FGD1 gene mutation
  • Primer and method for detecting FGD1 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It should be noted that the routine conditions and methods not described in the examples are generally used by experimenters in the field: for example, the fourth edition of the "Refined Molecular Biology Experiment Guide" edited by Osper and Kingston, Or follow the procedures and conditions recommended by the manufacturer.

[0031] A primer and method for detecting FGD1 659+27T>C and 482-113C>T site mutations. Increase primer, its base sequence is:

[0032] FGD1-EXON-3-F: TGTAAAACGACGGCCAGTGCCTCCTGAGTTCAAGCAAT

[0033] FGD1-EXON-3-R: AACAGCTATGACCATGTCTGAGGTGGGTGGTGGAC.

[0034] A kit for detecting FGD1 659+27T>C and 482-113C>T site mutations, comprising

[0035] (i) Blood DNA extraction reagents;

[0036] (ii) detection system PCR reaction solution;

[0037] (iii) Sequencing system reagents;

[0038] (iv) Positive control substance and negat...

Embodiment 2

[0043] The operation process of the blood genomic DNA extraction kit (Tiangen Biology):

[0044] (1) Genomic DNA extraction from blood

[0045] 1) Take 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 12,000rpm for 1min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed.

[0046] 2) Add 20 μl proteinase K solution and mix well.

[0047] 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0048] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0049] 5) Add the solution and floccul...

Embodiment 3

[0080] Take 20 cases of clinical samples, extract genomes, prepare reagents and detect according to the methods described in Examples 1 and 2. Add 1 μL of the sample to the PCR reaction solution of the detection system, and make a positive, negative, and blank control at the same time. Electrophoresis results such as figure 1 As shown, it shows that the primers of the present invention can effectively amplify blood samples, and the band is single.

[0081] After sequencing, a total of 8 samples were found to have FGD1 659+27T>C and 482-113C>T mutations, which were samples 1, 2, 6, 10, 11, 12, 13, and 18, and the two mutations occurred simultaneously in the samples. And all were homozygous mutations. The sequencing results of sample 1 (positive sample) and sample 3 (negative sample) are as follows Figure 2-5 shown. figure 2 and image 3 Sequencing screenshots of FGD1 659+27T site-negative and positive samples. Figure 4 and Figure 5 Sequencing screenshots of FGD1 482-...

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Abstract

The invention discloses a primer and method for detecting two base mutations of FGD1 659+27T>C and 482-113C>T; a Sanger sequencing technology is adopted and can be used for rapidly detecting the mutation sites. The results of detection completed by means of the primer and the method are accurate, and gene mutation of patients suffering from Aarskog syndrome can be assisted to be diagnosed. By means of the primer and method, whether or not generated gene mutation causes the Aarskog syndrome is judged and analyzed conveniently, and important reference significance is achieved for clinical differentiation and diagnosis on the Aarskog syndrome.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to primers for detecting FGD1 659+27T>C and 482-113C>T site mutations, which can be used to quickly detect FGD1 site mutations by using ordinary PCR combined with Sanger sequencing technology. Background technique [0002] Aarskog syndrome (Aarskog Syndrome, AAS) is a rare hereditary facial-digital-genital abnormality syndrome. Combined with multiple other abnormalities, the phenotype necessary for diagnosis is currently unclear. AAS is an X-linked recessive genetic disease caused by mutations in the FGD1 gene located at Xp11.21. [0003] The FGD1 gene (FYVE, RhoGEF and PH domain containing l, NC_000023) contains a total of 18 exons, which can encode a guanine nucleotide exchange factor (GEF). It has been found that a variety of FGD1 mutations can lead to the occurrence of AAS, including deletion mutations in exons 4, 6, 9-12 and 17, insertion mutatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/6869C12Q2600/156C12Q2535/101C12Q2531/113
Inventor 牛林梅吴鹏飞王淑一
Owner 杭州艾迪康医学检验中心有限公司
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