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Primers and method for detecting mutation of GNAS genetic locus

A technology of gene loci and sequencing primers, which is applied in the fields of life science and biology, can solve problems such as unclear functions, and achieve the effects of difficult detection, high cost, and reduced cost and difficulty

Inactive Publication Date: 2018-04-13
南昌艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Overall, activating mutations in GNAS can modify cell growth and may be oncogenic, however, the role of GNAS as an oncogene is still unclear, so GNAS gene mutation detection is crucial for the diagnosis, identification and prevention of tumors

Method used

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  • Primers and method for detecting mutation of GNAS genetic locus
  • Primers and method for detecting mutation of GNAS genetic locus
  • Primers and method for detecting mutation of GNAS genetic locus

Examples

Experimental program
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Embodiment 1

[0028] 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.

[0029] A primer for detecting mutations in GNAS gene sites. The primers are designed for mutation hotspots of exon 5 12384GCC>GCT and 12450ATC>ATT sites, including:

[0030] The primers for amplifying the hotspot region of GNAS gene mutation, its base sequence is:

[0031] GNAS-EXON-5-F: TGTAAAACGACGGCCAGTGCAGTGAGAAGGCAACCAA

[0032] GNAS-EXON-5-R: AACAGCTATGACCATGGGCTGTCACTCATGTTCCTAT

[0033] A test kit for detecting mutations in GNAS gene sites, comprising

[0034] (i) Blood DNA extraction reagents;

[0035] (ii) detecti...

Embodiment 2

[0042] The operation process of the blood genomic DNA extraction kit (Tiangen Biology):

[0043] (1) Genomic DNA extraction from blood

[0044] 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.

[0045] 2) Add 20 μl proteinase K solution and mix well.

[0046] 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.

[0047] 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.

[0048] 5) Add the solution and floccul...

Embodiment 3

[0076] Fifteen clinical samples were taken, and the genome was extracted, reagents were prepared and tested according to the methods described in Examples 1 and 2. Add 1 μl of the sample to the detection system PCR reaction solution. Electrophoresis results such as figure 2 As shown, it shows that the primers of the present invention can effectively amplify blood samples, and the band is single.

[0077] The test results of the samples were image 3 and Figure 4 shown. image 3 and Figure 4 Sequencing screenshots of the negative and positive samples at base 12384 of exon 5 of the GNAS gene. Figure 5 and Image 6 Sequencing screenshots of the negative and positive samples at base 12,450 of exon 5 of the GNAS gene.

[0078] It can be seen from the detection results that the primers of the present invention have included the sequence of the hotspot region, and can amplify exon 5 of the GNAS gene, and the sequencing results are completely accurate. Among them, there ar...

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Abstract

The invention discloses primers and a method for detecting mutation of GNAS basic group. The primers comprise hotspot mutation regions of No.5 exons of a GNAS gene; a Sanger sequencing technique is adopted to detect mutation locus quickly. A detection result obtained by the invention is accurate and can assist in diagnosing mutation conditions in the hotspot regions. By utilizing the primers and the method disclosed by the invention, the mutation of the No.5 exons 12384GCC greater than GCT and 12450ATC greater than ATT of the GNAS gene are found for the first time; the discovery of the mutation loci favorably judges and analyzes whether the generated gene mutation can cause related disease or not and assists in screening the cause of diseases.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to primers for detecting mutations of GNAS gene sites, which can be used for rapid detection of mutations of GNAS sites by adopting common PCR technology. Background technique [0002] The GNAS gene is located on chromosome 20, consists of 13 exons and 12 introns, and encodes the alpha subunit (Gsα) of the guanine nucleotide binding protein (G protein). Gsα forms heterotrimeric G protein complexes with β and γ subunits that mediate signal transduction from a large number of hormone and growth factor-activated transmembrane receptors to a variety of intracellular signaling pathways. Ligand-bound G protein-coupled receptors activate the Gs protein by promoting GDP exchange of Gs on Gsα, resulting in dissociation from the receptor and the βγ-complex. The free Gsα subunit interacts with adenylyl cyclase to stimulate cAMP synthesis until hydrolysis of GTP convert...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2535/122
Inventor 黄开新吴鹏飞王淑一
Owner 南昌艾迪康医学检验实验室有限公司
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