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Method and primer for detecting 8th and 25th whole exons of TEX11 gene

A technology of TEX11S-511-F and TEX11S-511-R, which is applied to the primers for the mutation of the whole exon 25 to detect the 8th field of the TEX11 gene, can solve the problems of high mutation rate, multiple mutation types, and prevention of in-depth research , to achieve the effect of low cost, simple operation and high sensitivity

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

Due to the large capacity of the TEX11 gene, multiple types of mutations, high mutation rate, and scattered distribution of mutation sites, in-depth research on the mutation of the TEX11 gene is prevented, and the fluorescent quantitative PCR method is not applicable.

Method used

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  • Method and primer for detecting 8th and 25th whole exons of TEX11 gene
  • Method and primer for detecting 8th and 25th whole exons of TEX11 gene
  • Method and primer for detecting 8th and 25th whole exons of TEX11 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Primers for detection of exons 8 and 25 of the TEX11 gene, including: 2 pairs of forward and reverse primers for amplifying and covering the entire exons 8 and 25 of the TEX11 gene; wherein amplifying the No. 8 TEX11 gene Because of TEX11-511-F and TEX11-511-R of the whole exon, amplify the No. 25 whole exon of the TEX11 gene because of TEX11-2092-F and TEX11-2092-R, the base of the primer The base sequence is:

[0058] TEX11-511-F: 5'CCTTAGCCACCCATAGA3'

[0059] TEX11-511-R: 5'ATGCTAACTGTTGCTTTT3'

[0060] TEX11-2092-F: 5'ATCAGGGAAGGCAAACAT3'

[0061] TEX11-2092-R: 5'AGAAATACAAGGGCGAAT3'

[0062] In the detection, first use the above two pairs of forward and reverse primers to amplify the DNA fragments covering and detecting the whole exons No. TEX11-511-R sequenced the amplified product of the No. 8 whole exon of the TEX11 gene, and used the sequencing primers TEX11-2092-F and TEX11-2092-R to sequence the No. 25 whole exon of the TEX11 gene The amplified products...

Embodiment 2

[0084] The operation process of the blood DNA extraction kit (Tiangen Biology):

[0085] (1) Genomic DNA extracted from blood:

[0086] 1) Take 500uL of blood and add 1000uL of red blood cell lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 3000rpm for 5min, suck off the supernatant, leave the white blood cell pellet, add 200uL buffer GA, shake until thoroughly mixed.

[0087] 2) Add 20 μl proteinase K solution and mix well.

[0088] 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.

[0089] 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.

[0090] 5) Put the solution and flocculent prec...

Embodiment 3

[0115] Take 24 samples from patients with clinical azoospermia, and detect whether there is TEX11 gene mutation in the 24 samples, sample number 1-24. The genome was extracted, reagents were prepared and tested according to the method described in Example 2. Add 1 μl of each sample to the detection system PCR reaction solution. At the same time, make positive, negative, and blank controls. Detect with common PCR instrument, the time is 160 minutes.

[0116] Electrophoresis results such as figure 2 , 3 , 4, and 5 show that the primers TEX11-511-F / R and TEX112092-F / R of the present invention can effectively amplify blood samples with a single band.

[0117] Part of the forward sequencing results of the No. 8 whole exon sequence of sample 3 are as follows: Figure 6 As shown, it is wild type, and no TEX11 mutation was detected.

[0118] Partial forward sequencing results of No. 8 full exon sequence of sample 5 are as follows: Figure 7 As shown, it is wild type, and no TE...

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Abstract

The invention discloses a method, a primer and a kit for detecting 8th and 25th whole exons of a TEX11 gene, wherein the primer and the kit respectively comprise two pairs of forward and reverse primers and two pairs of sequencing primers which are used for amplifying and covering sequences of the 8th and 25th whole exons of the TEX11 gene. On the basis of adoption of a Sanger sequencing method, the two pairs of sequencing primers can be utilized for quickly detecting mutation condition of the sequences of the 8th and 25th whole exons of the TEX11 gene of a patient with azoospermia, wherein a main mutation site M171V on the 8th exon and a main mutation site A698T on the 25th exon are contained. The primer and the method which are disclosed by the invention are accurate and efficient in detection.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a primer and a method for detecting mutations of No. 8 and No. 25 whole exons of TEX11 gene. Background technique [0002] Azoospermia (Azoospermia) refers to multiple semen examinations (generally more than 3 times) but no sperm is found. Clinically, according to whether there is obstruction of the vas deferens, azoospermia is divided into two types: obstructive azoospermia and non-obstructive azoospermia. The former means that the testis can produce sperm, but the vas deferens is blocked and the sperm cannot be discharged smoothly. The latter is when the testicles fail to produce or produce very little sperm. The causes of non-obstructive azoospermia are complex, and chromosomal abnormalities are one of the causes of this genetic disease. Chromosomal abnormalities can be detected in 20% of non-preventive azoospermic men, including azoospermic factors...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6869C12Q2600/156
Inventor 刘赵玲林筱剑王淑一
Owner 杭州艾迪康医学检验中心有限公司
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