Primers and method for detecting polymorphic hotspot mutation condition of DOCK2 gene

A technology of polymorphic hotspots and sequencing primers, applied in the fields of life sciences and biology, can solve the problems of lack of resistance, deficiency, and inability to synthesize immunoglobulins, etc., and achieve the effect of difficult detection, high cost, and reduced cost and difficulty

Inactive Publication Date: 2016-04-20
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

Combined immunodeficiency disease will lead to congenital abnormal differentiation of lymphoid stem cells, and the baby will lack T cells and B cells after birth, so that both humoral immunity and cellular immunit

Method used

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  • Primers and method for detecting polymorphic hotspot mutation condition of DOCK2 gene
  • Primers and method for detecting polymorphic hotspot mutation condition of DOCK2 gene
  • Primers and method for detecting polymorphic hotspot mutation condition of DOCK2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] A primer for detecting polymorphic mutation sites of the DOCK2 gene, the design of the primers is specific amplification primers designed for DOCK2 mutation hotspots, including:

[0079] Primers for amplifying DOCK2 gene sequences containing exons 6, 22, 33, 37, 40, and 44, the base sequences of which are:

[0080] DOCK2-1F: TGTAAAACGACGGCCAGTTTATGGGTTGTTTCTTGTCTCCT

[0081] DOCK2-1R: AACAGCTATGACCATGCCATCGGAAGTAGGTTAAATGAG

[0082] DOCK2-2F: TGTAAAACGACGGCCAGTCTTTAACCTTTGATTGAATGCCT

[0083] DOCK2-2R: AACAGCTATGACCATGTAGGTTGGATTCTTGAGCTGTTT

[0084] DOCK2-3F: TGTAAAACGACGGCCAGTCGTCGCCGAACTGTTTTAT

[0085] DOCK2-3R: AACAGCTATGACCATGGAGAAAGAGGCTAAGCAAATACA

[0086] DOCK2-4F: TGTAAAACGACGGCCAGTGTTATCCAGGGAGGAGGACTTT

[0087] DOCK2-4R: AACAGCTATGACCATGCTCTTCAACTTGCTGTGAGGC

[0088] DOCK2-5F: TGTAAAACGACGGCCAGTGCTTACTGGCTGCTTTACGAG

[0089] DOCK2-5R: AACAGCTATGACCATGTATGATGAGGGCTATTGACTGG

[0090] DOCK2-6F: TGTAAAACGACGGCCAGTTTGCAATATTTATTCTGCTTTCG

[0091] DOCK2-6...

Embodiment 2

[0100] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0101] (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 ...

Embodiment 3

[0128] 16 cases of clinical samples (sample number 1-16) were taken according to the reagents and methods of Examples 1 and 2 to extract genome, prepare reagents, amplify and sequence. Add 1 μl of each sample to the detection system PCR reaction solution. Electrophoresis results such as figure 2 , 3 , 4, 5, 6, and 7, indicating that the primers DOCK2-1F / R, DOCK2-2F / R, DOCK2-3F / R, DOCK2-4F / R, DOCK2-5F / R, DOCK2-6F of the present invention / R can effectively amplify blood samples with a single band.

[0129] The test results of samples 1, 2, 3, 4, 5, and 6 are as follows Figure 8 , 9 , 10, 11, 12, and 13:

[0130] Figure 8 It shows the wild-type sequencing screenshot of DOCK exon 26 of sample 1, indicating that exon 6 of sample 1 is not mutated.

[0131] Figure 9 It shows the wild-type sequencing screenshot of exon 222 of DOCK of sample 2, indicating that exon 22 of sample 2 is not mutated.

[0132] Figure 10 It shows the wild-type sequencing screenshot of DOCK233 ...

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Abstract

The invention discloses primers and a method for detecting myelosis myelodysplastic syndrome and especially detecting DOCK2 gene mutation of a patient with myelosis myelodysplastic syndrome. The primers comprise (i) primers for amplifying 6th, 22nd, 33rd, 37th, 40th and 44th exon sequences of the DOCK2 gene. An Sanger sequencing technique and sequencing primers are adopted. The primers and method can be used for quickly detecting mutation of 6th, 22nd, 33rd, 37th, 40th and 44th exons of the DOCK2 gene in the body of a patient with myelosis myelodysplastic syndrome. The primers and method have the advantage of accurate detection result, can be used for assisted diagnosis of severe combined immunodeficiency disease, and have important reference meanings for early intervention and early therapy.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a primer and a method for detecting polymorphic hotspot mutations of DOCK2 gene. Background technique [0002] Combined immunodeficiency disease is a polygenic genetic disease, which can be divided into: X-linked inheritance, autosomal recessive inheritance and sporadic. Combined immunodeficiency disease will lead to congenital abnormal differentiation of lymphoid stem cells, and the baby will lack T cells and B cells after birth, so that both humoral immunity and cellular immunity will be deficient, and it will not be able to synthesize its own immunoglobulin, which will lead to the patient's resistance to bacteria, fungi, Lack of resistance to viruses, etc., prone to invasive infections. Children with onset within 1-2 months after birth, if not effectively treated, more than 1 year old died. At present, the disease has been listed as an "emergency case...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/686C12Q2600/156C12Q2527/143C12Q2527/101
Inventor 林筱剑林有升王淑一
Owner 杭州艾迪康医学检验中心有限公司
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