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A mitochondrial DNA SNP marker associated with noa of unknown clinical cause and its application

A marker, unknown technology used in genetic engineering and reproductive medicine

Active Publication Date: 2014-10-22
夏彦恺
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of mitochondrial DNA genetic variation in the diagnosis of NOA. If the mitochondrial DNA genetic variation of NOA susceptibility can be screened out as a biomarker, and the corresponding diagnostic kit can be developed, it will be a powerful step in the diagnosis of NOA. The promotion of the drug has also opened up new ways for its drug screening, drug efficacy evaluation and targeted therapy

Method used

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  • A mitochondrial DNA SNP marker associated with noa of unknown clinical cause and its application
  • A mitochondrial DNA SNP marker associated with noa of unknown clinical cause and its application
  • A mitochondrial DNA SNP marker associated with noa of unknown clinical cause and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: collection of samples and arrangement of sample data

[0082] The inventor collected a large number of blood samples of NOA patients from the Reproductive Medicine Center of Nanjing Medical University from June, April, 2007 to January, 2011. After sorting out the sample data, the inventor selected 1409 samples that met the following criteria Experimental samples for genome-wide microarray scanning and single SNP TaqMan genotyping:

[0083] 1. Repeated semen quality test / testicular biopsy, diagnosed as azoospermia;

[0084] 2. The semen of one ejaculation was centrifuged without spermatozoa, and obstructive etiology was ruled out;

[0085]3. Sexual function is normal. Exclude patients with known causes such as cryptorchidism, history of vascular trauma, orchitis, vas deferens obstruction, vasectomy, polychromosomal abnormalities, and Y chromosome azoospermia factor microdeletion;

[0086] 4. Healthy male controls matched with the age of the cases.

[0087]...

Embodiment 2

[0088] Whole Genome Scanning of SNP in Example 2 Peripheral Blood DNA

[0089] Among the above-mentioned eligible 192 patients with NOA of unknown clinical cause and 192 healthy male controls, the two groups were age-matched. The two groups of people were sequenced by Illumina to obtain relevant results. The specific steps are:

[0090] 1. Add hemolysis reagent (i.e. lysate, 40 parts) to the peripheral blood stored in a 2ml cryopreservation tube. Dilute the TrisHcl solution to 2000ml, the same below), mix it upside down and transfer it completely;

[0091] 2. Removal of red blood cells: Fill the 5ml centrifuge tube to 4ml with hemolysis reagent, mix by inverting, centrifuge at 4000rpm for 10min, and discard the supernatant. Add 4ml of hemolysis reagent to the precipitate, invert and wash again, centrifuge at 4000rpm for 10min, and discard the supernatant;

[0092] 3. Extract DNA: Add 1ml of extract solution to the precipitate (each 300ml contains 122.5ml0.2M sodium chlorid...

Embodiment 3

[0101] Example 3 TaqMan Genotyping of Single Genetic Variations

[0102] 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 536 cases of NOA of clinically unknown cause and 489 healthy male controls. The specific steps were as follows:

[0103] 1. Add the hemolysis reagent to the peripheral blood stored in the 2ml cryopreservation tube, mix it upside down and transfer it completely;

[0104] 2. Removal of red blood cells: Fill the 5ml centrifuge tube to 4ml with hemolysis reagent, mix by inverting, centrifuge at 4000rpm for 10min, and discard the supernatant. Add 4ml of hemolysis reagent to the precipitate, invert and wash again, centrifuge at 4000rpm for 10min, and discard the supernatant;

[0105] 3. Extract DNA: Add 1ml of extract solution and 8μl of proteinase K to the precipitate, fully oscillate and mix on the shaker, and bathe overnight at 37°C;

[0106] 4. Remove protein: add 1ml of...

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Abstract

The invention relates to a clinically unexplained NOA (non-obstructive azoospermia)-related mitochondrial DNA SNP (single nucleotide polymorphism) marker and applications thereof, belonging to the fields of genetic engineering and reproductive medicine. The marker is a combination of the following mitochondrial DNA SNP sites: T3394C, A6881G, G11696A, G13135A, G13928C, A15662G, T15968C, T16224C and G16319A. The marker and specific primers and / or specific probes thereof can be used for auxiliary diagnosis of the clinically unexplained NOA, greatly improving the sensitivity and specificity of diagnosis.

Description

field of invention [0001] The invention belongs to the field of genetic engineering and reproductive medicine, and relates to a mitochondrial DNA SNP marker related to NOA (non-obstructive azoospermia) of unknown clinical cause and application thereof. Background technique [0002] About 8-15% of the couples of childbearing age in the world have different degrees of fertility obstacles, of which the male factor accounts for about 50%. Studies have shown that the quality of human semen has declined significantly for more than half a century. It is currently believed that the occurrence of spermatogenesis disorders is a multi-factor and multi-stage process, which is the result of the joint action of environmental risk factors (external factors) and individual genetic factors (internal factors). . Among them, individual genetic factors include changes in chromosomal genetic factors, abnormal epigenetic modifications, and changes in the genetic structure of the mitochondrial ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 夏彦恺陆春城吴炜秦玉峰杜桂珍许妙斐王心如
Owner 夏彦恺
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