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SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof

An asthenospermia and marker technology, applied in the fields of genetic engineering and reproductive medicine

Active Publication Date: 2014-05-07
NANJING MEDICAL UNIV
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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 asthenospermia. If the mitochondrial DNA genetic variation susceptible to asthenospermia can be screened out as a biomarker, and the corresponding diagnostic kits can be developed, the diagnosis of asthenospermia will be greatly improved. The status quo will definitely be a powerful impetus, and it will also open up new ways for drug screening, drug efficacy evaluation and targeted therapy

Method used

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  • SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof
  • SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof
  • SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 The collection of samples and the arrangement of sample data

[0065] The inventor collected a large number of blood samples from patients with asthenozoospermia of unknown clinical cause from June 4, 2007 to January 2011, and selected 1724 Samples that meet the following criteria for whole-genome microarray scanning and single-SNP SNaPshot genotyping experimental samples:

[0066] 1. Repeated semen quality test, diagnosed as asthenozoospermia;

[0067] 2. Normal sexual function; exclude patients with known etiologies such as cryptorchidism, orchitis, vas deferens obstruction, vasectomy, polychromosomal abnormalities, and microdeletions of Y chromosome azoospermia factor;

[0068] 3. Healthy male controls matched with the age of the cases.

[0069] The demographic data and clinical data of these samples were collected systematically.

Embodiment 2

[0070] Example 2 Whole Genome Scanning of SNPs in Peripheral Blood DNA

[0071] Among the above-mentioned eligible 236 patients with asthenozoospermia of unknown clinical cause and 234 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:

[0072] 1. Add hemolysis reagent (that is, lysate, 40 parts) to the blood cells stored in the 2ml cryopreservation tube. Dilute to 2000ml, the same below), invert and mix completely before transferring.

[0073] 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.

[0074] 3. Extract DNA: add 1ml of extract solution to the precipitate (each 300ml contains 122.5ml 0.2M sodium c...

Embodiment 3

[0084] Example 3 SNaPshot Genotyping of Single Genetic Variations

[0085] The SNPs found to be associated with the onset of clinically unexplained asthenozoospermia in the above genome-wide scan were detected in another 688 clinically unexplained asthenozoospermia cases and 566 healthy male controls. The specific steps were as follows:

[0086] 1. Add the hemolysis reagent to the blood cells stored in the 2ml cryopreservation tube, mix it upside down and transfer it completely.

[0087] 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.

[0088] 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.

[0089] 4. Remove...

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Abstract

The invention belongs to the fields of genetic engineering and reproductive medicine and discloses an SNP marker of mitochondria DNA related to asthenospermia with unknown clinical causes and application thereof. The marker is a combination of C5601T, T12338C, Al2361G, G13928C, Al5851G, C16179T and G16291A, can be used for preparing an auxiliary diagnosis kit for asthenospermia with unknown clinical causes and has high specificity and sensitivity.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to a mitochondrial DNA SNP marker related to asthenospermia 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 male infertility 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 modification, and genetic structure variation in mitochondrial genome. Mitochondrial genome is the onl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 陆春城王嵘夏彦恺吴炜许妙斐秦玉峰陈敏健杜桂珍王心如
Owner NANJING MEDICAL UNIV
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