Primers and method for detecting gene mutation of JAK3 gene intron 2

An intron and sequencing primer technology, applied in the fields of life science and biology, can solve the problem of the loss of kinase activity, and achieve the effect of reducing cost and difficulty, high cost and difficult detection.

Pending Publication Date: 2019-05-21
合肥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

JH2 is a pseudo-kinase domain. Although it lacks catalytic activity, it has some necessary regulatory functions. Many patients or artificial mutations in this region will lead to loss of kinase activity.
The function of the JH3-JH5 region has not yet been clarified, and it may be related to the assembly of JAKs in vivo

Method used

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  • Primers and method for detecting gene mutation of JAK3 gene intron 2
  • Primers and method for detecting gene mutation of JAK3 gene intron 2
  • Primers and method for detecting gene mutation of JAK3 gene intron 2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A primer for detecting the mutation of the JAK3 gene intron 2 gene site, the design of the primer is an amplification primer designed for the JAK3 gene intron 2, including:

[0044] Amplify the primer of JAK3 gene intron 2, its base sequence is:

[0045] JAK3-Int 2-F: TGTAAAACGACGGCCAGTTTACAGGCATAAGCCACCG

[0046] JAK3-Int 2-R: AACAGCTATGACCATGACACCCTTCTCCATTACAAAA.

[0047] A test kit for detecting JAK3 gene intron 2 site mutation, comprising

[0048] (i) Blood DNA extraction reagents;

[0049] (ii) detection system PCR reaction solution;

[0050] (iii) Sequencing system reagents;

[0051] Among them, the tissue DNA extraction reagent can be purchased from commercial reagents such as Tiangen DNA extraction kit.

[0052] Detection system PCR amplification reaction liquid includes: 2×PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U / μl); JAK3 gene intron 2 upstream and downstream primers The concentration of primers JAK3-Int 2-F / R is 10 μM.

[0053] Sequencing syst...

Embodiment 2

[0055] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0056](1) Extract tissue DNA from blood: 1) Extract 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inverting, and place 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. 2) Add 20 μl proteinase K solution and mix well. 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. 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. 5) Add the solution and flocculent precipitate obtained in the previous step i...

Embodiment 3

[0082] The clinical samples of 5 cases were extracted genome, prepared reagents, amplified and sequenced according to the reagents and methods of Examples 1 and 2. Add 1 μl of sample to each detection system PCR reaction solution. Electrophoresis results such as figure 1 As shown, it shows that the primer JAK3-Int 2-F / R of the present invention can effectively amplify the blood sample, and the band is single.

[0083] The test results of the samples were figure 2 , 3 , 4, 5, 6, and 7 show:

[0084] figure 2 It shows the sequence screenshots of JAK3 gene intron 2 mutant type 3821C>T site of samples 1, 3 and 4, indicating that 3821C>T mutation occurred in intron 2 of samples 1, 3 and 4.

[0085] image 3 It shows that the intron 2 mutations of the JAK3 gene of samples 1, 3, and 4 are 3839G>A and 3841G>A site sequencing screenshots, indicating that the intron 2 of samples 1, 3, and 4 has 3839G>A and 3841G>A mutations .

[0086] Figure 4 It shows the sequence screensho...

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Abstract

The invention discloses primers and method for detecting the mutation of loci where 3821C is larger than T, 3839G is larger than A, 3841G is larger than A and 3895C is larger than T in the region of aJAK3 gene intron 2. The primers include (i) the primers for amplifying the sequence of the JAK3 gene intron 2, and the Sanger sequencing technology and the sequencing primers are adopted. By means ofthe Sanger sequencing technology, the mutant loci can be rapidly found. The method is accurate in detection result and can assist in detecting the mutation conditions of the region. The mutation of the loci where 3821C is larger than T, 3839G is larger than A, 3841G is larger than A and 3895C is larger than T in the region of the JAK3 gene intron 2 can be rapidly detected. The method is accuratein detection result and has reference significance in exploring pathogenesis of diseases.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a primer and a detection method for detecting the mutation of JAK3 gene intron 2, which can be used to quickly detect the mutation of JAK3 gene intron 2 by adopting common PCR technology. Background technique [0002] The JAK3 gene is located on chromosome 19 p12.13.1, its open reading frame has 3372 bases, encodes 1124 amino acids, and has a total of 24 exons. From the carboxyl group to the amino acid terminal are JH1, JH2, JH3, JH4, JH5, JH6, JH7, respectively. JH1 is a kinase domain and plays an important role in regulating kinase activity. JH2 is a pseudo-kinase domain. Although it lacks catalytic activity, it has some necessary regulatory functions. Many patients or artificial mutations in this region will lead to loss of kinase activity. The function of the JH3-JH5 region has not been studied clearly, and it may be related to the assembly of JAKs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
Inventor 刘赵玲吴鹏飞王淑一
Owner 合肥艾迪康医学检验实验室有限公司
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