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Single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of SNP marker

A technique for assisting diagnosis of azoospermia, applied in the field of SNP markers

Active Publication Date: 2012-04-04
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the application of SNP in the diagnosis of NOA of unknown clinical cause. If the SNP of NOA susceptibility of unknown clinical cause can be screened out as a biomarker and a corresponding diagnostic kit can be developed, it will be of great help to the diagnosis of NOA with unknown clinical cause in my country. The diagnosis status of NOA will definitely be a powerful impetus, and it will also open up new ways for its drug screening, drug efficacy evaluation and targeted therapy

Method used

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  • Single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of SNP marker
  • Single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of SNP marker
  • Single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of SNP marker

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] The collection of embodiment 1 sample and the arrangement of sample data

[0071] The inventor collected a large number of blood samples of NOA patients from the Reproductive Medicine Center of Nanjing Medical University from April 2005 to January 2011. After sorting out the sample data, the inventor selected 4,700 samples that meet the following criteria. Whole genome Experimental samples for microarray scanning and single SNP TaqMan genotyping:

[0072] 1. Repeated semen quality test / testicular biopsy, diagnosed as azoospermia;

[0073] 2. The semen of one ejaculation was centrifuged without sperm, and the cause of obstruction was ruled out;

[0074] 3. Sexual function is normal; patients with known etiologies such as cryptorchidism, history of vascular trauma, orchitis, vas deferens obstruction, vasectomy, polychromosomal abnormalities, and Y chromosome azoospermia factor microdeletion are excluded

[0075] 4. Healthy male controls matched with the age of the case ...

Embodiment 2

[0077] Whole Genome Scanning of SNP in Example 2 Peripheral Blood DNA

[0078] Among the above-mentioned eligible 1000 patients with NOA of unknown clinical cause and 1700 healthy male controls, the two groups were age-matched. The two groups of people were detected by Affymetrix6.0 chip to obtain relevant results. The specific steps are:

[0079] 1. Add the hemolysis reagent to the leukocytes stored in the 2ml cryopreservation tube, mix it upside down and transfer it completely.

[0080] 2. Removal of red blood cells: Fill the 5ml centrifuge tube to 4ml with hemolysis reagent, mix by inverting, centrifuge at 4000rpm for 10 minutes, and discard the supernatant. Add 4ml of hemolysis reagent to the precipitate, invert and wash again, centrifuge at 4000rpm for 10 minutes, and discard the supernatant.

[0081] 3. Extract DNA: Add 1ml extract solution to the precipitate (each 300ml contains 122.5ml 0.2M sodium chloride, 14.4ml 0.5M ethylenediaminetetraacetic acid, 15ml 10% sodiu...

Embodiment 3

[0091] TaqMan Genotyping of Example 3 Single SNP

[0092] The SNPs found to be associated with the onset of NOA of clinically unknown cause by the above genome-wide scan were detected in another 1000 cases of NOA of clinically unknown cause and 1000 healthy male controls. The specific steps were as follows:

[0093] 1. Add the hemolysis reagent to the leukocytes stored in the 2ml cryopreservation tube, mix it upside down and transfer it completely.

[0094]2. Removal of red blood cells: Fill the 5ml centrifuge tube to 4ml with hemolysis reagent, mix by inverting, centrifuge at 4000rpm for 10 minutes, and discard the supernatant. Add 4ml of hemolysis reagent to the precipitate, invert and wash again, centrifuge at 4000rpm for 10 minutes, and discard the supernatant.

[0095] 3. Extract DNA: Add 1ml of extract solution and 8μl of proteinase K to the precipitate, fully oscillate and mix on a shaker, and bathe overnight at 37°C.

[0096] 4. Remove protein: add 1ml of saturated p...

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Abstract

The invention belongs to the fields of gene engineering and reproductive medicine, and discloses a single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of the SNP marker. The marker is a combination of rs1014331, rs10765875, rs10889203, rs11066453, rs11167809, rs12097821, rs1990264, rs3000811, rs3130048, rs391129, rs4716515, rs658239, rs6589102, rs7042232, rs7099208, rs7144703, rs7194 and rs985421. The marker can be used for preparing a clinically cryptogenic non-obstructive azoospermia aided diagnosis kit.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to a SNP marker related to the auxiliary diagnosis of non-obstructive azoospermia of unknown clinical cause and its application. Background technique [0002] Fertility is a complex process that cannot be completed by a single individual, but depends on both men and women, and can only be successfully completed when various conditions are suitable for both parties. According to WHO statistics, about one out of every seven couples has various degrees of fertility obstacles, and nearly half of them are related to the male factor, which is called male infertility. Male infertility has caused heavy mental and economic burdens to individuals and families, and has become one of the unstable factors of families and society. Therefore, male infertility has gradually become a global public social problem, and it is one of the important contents of reproductive ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 沙家豪胡志斌沈洪兵周作民董静
Owner NANJING MEDICAL UNIV
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