Detection method for Y chromosome micro-deleted gene

A technology of Y chromosome and detection method, applied in the field of genetic engineering, can solve the problems of increasing the cost of reagents, unable to completely detect AZF fragments, etc., and achieve the effects of reducing the formation of primer-dimers, facilitating clinical experiment operations, and simplifying PCR reaction conditions.

Inactive Publication Date: 2009-11-11
成都军区昆明总医院
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

This results in the inability to fully detect the micro-deletion of the AZF fragment (Chinese invention pate...

Method used

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  • Detection method for Y chromosome micro-deleted gene
  • Detection method for Y chromosome micro-deleted gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Human Genomic DNA Extraction—Extract DNA from 250ul of anticoagulated whole blood by conventional boiling lysis method or column elution method, and the expected DNA yield is about 4-12ug, which can be used as template DNA.

[0058] PCR amplification - multiplex PCR amplification is divided into tube A and tube B. The 30ul PCR reaction system is: 1×PCR buffer (50mmol / L KCl, 10mmol / L Tris-HCl, pH 9.0), 1.5mMMgCl 2 , 200uM dNTP, 5~10ng template DNA, and 1U Taq DNA polymerase (Promega); the concentrations of 5 pairs of primers added to tube A are: MSRY 0.12uM, MsY254 0.1uM, MsY127 0.05uM, MsY86 0.1uM, YUP 0.4uM; The concentrations of 5 pairs of primers added to tube B are: MSRY 0.1uM, MsY1340.12uM, MsY84 0.12uM, MsY255 0.05uM, YUP 0.4uM, and the reaction was carried out in a 9700 PCR reaction instrument of ABI Company. A positive control (genome DNA of normal fertile male), a negative control (genome DNA of female) and a water blank control were set up for each test.

[...

Embodiment 2

[0068] The selection of the STS site of the AZF fragment was based on the standards recommended by the "European Y Chromosome Microdeletion Molecular Diagnosis Guidelines 2004 Edition" issued by the European Association of Andrology EAA (clinical level) and the European Molecular Genetic Laboratory Quality Control Association EMQN (specific experimental operation level) performed (SIMONI M., et al. 2004, International Journal of Andrology, 27, 240-249). The AZFa region selects the sY84 and sY86 sites:

[0069] The base sequence of the primer for specifically amplifying AZFa region sY84 is:

[0070] sY84-F: 5’-AGA AGG GTC TGA AAG CAG GT-3’

[0071] sY84-R: 5'-GCC TAC TAC CTG GAG GCT TC-3'

[0072] The base sequence of the primer for specifically amplifying sY86 in the AZFa region is:

[0073] sY86-F: 5'-AGA CTA TGC TTC AGC AGG TC-3'

[0074] sY86-R: 5’-GAA CCG TAT CTA CCA AAG CAG C-3’

[0075] AZFb region selects sY127 and sY134 sites:

[0076] The base sequence of the pr...

Embodiment 3

[0101] Genomic DNA was extracted from human peripheral blood. For PCR reaction, multiplex PCR takes a 15ul PCR reaction system as an example. The reaction system (pH 8.5) includes the following reagents: PCR reaction solution, Taq enzyme, dATP, dGTP, dCTP, dTTP, MgCl 2 , primers, template DNA. The final concentration of dNTP reaction is 200uM; MgCl 2 The final concentration of the reaction was 1.5 mM. The PCR amplification was divided into two tubes: tube A with 5 pairs of primers for composite amplification; tube B with 5 pairs of primers for multiplex amplification, and both tubes were designed with MSRY primers as internal controls. Primer pairs are grouped as follows:

[0102] Group A: MSRY, MsY254(AZFc), MsY127(AZFb), MsY86(AZFa), YUP

[0103] Group B: MSRY, MsY134(AZFb), MsY84(AZFa), MsY255(AZFc), YUP

[0104] PCR cycle conditions: pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 40 s, annealing at 54°C for 40 s, extension at 72°C for 40 s, after 3...

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Abstract

The invention discloses a detection method for Y chromosome micro-deleted gene. In the method, after human peripheral blood genome DNA is extracted, multiple PCR primer design is used, wherein a multiple PCR reaction system comprises N pairs of chimeric primers for specifically amplifying the STSs site in an AZF segment of a Y chromosome and one pair of universal primers. N+1 pairs of primers are participated in the multiple PCR reaction, and the reactions of DNA template such as denaturalization, anneal and extending in circulating amplification target are carried out in a same reaction system; 10ulPCR products are taken and dye by EB, and electrophoresis is carried out through 3% agarose. The electrophoresis voltage is 4 V/cm, and the electrophoresis time is 20 minutes; and then the electrophoretogram is observed under an ultraviolet transilluminator so that the result is judged. Therefore, a detection method with simplified PCR reaction conditions, high amplification efficiency and easy operation for clinic experiments is established.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically, the invention relates to a method for detecting Y chromosome microdeletion. Background technique [0002] According to the research report of the World Health Organization (WHO) in 1997, infertile couples account for 15% of married couples of childbearing age in developed countries, and male infertility accounts for 50% of the total number of infertile couples. For the 50% of male infertility patients, according to clinical statistics, the etiology of their infertility can be roughly divided into the following four categories: spermatogenesis disorder, vas deferens obstruction, gonad accessory abnormality, and sexual function abnormality. Among them, spermatogenesis disorder is the most common. Factors causing spermatogenesis disorders mainly include two aspects: genetic factors and non-genetic factors. The rate of infertility caused by genetic factors accounts for abou...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 叶峻杰郭海李宗芳王跃力阎慧李江川途昕明王磊张雨龙沈君
Owner 成都军区昆明总医院
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