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Y chromosome microdeletion diagnosis and method for carrying out PCR amplification

A technology of Y chromosome and diagnostic reagents, which is applied in the field of reagents for diagnosing subtle deletions of Y chromosomes, and can solve the problems of large Y chromosomes and lack of stable molecular genetic testing reagents, etc.

Inactive Publication Date: 2005-07-06
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, domestic laboratories can detect large Y chromosome deletions with cytogenetic methods, but there are no stable molecular genetic detection reagents

Method used

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  • Y chromosome microdeletion diagnosis and method for carrying out PCR amplification
  • Y chromosome microdeletion diagnosis and method for carrying out PCR amplification
  • Y chromosome microdeletion diagnosis and method for carrying out PCR amplification

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specific Embodiment approach 1

[0005] Specific embodiment 1: The diagnostic reagent for fine deletion of Y chromosome of the present embodiment is made of the following components: (1) Dissolve the dry powder of the following primers respectively with TE of pH=7~8: sY87-F, sY87-R, sY143-R , ex2DAZ-F, ex2DAZ-R, RhEY-F, RhEY-R, respectively prepared sY87-F, sY87-R, sY143-R, ex2DAZ-F, ex2DAZ-R, RhEY-F, RhEY- R solution; (2) Add the following components to the 24 μl reaction system: 0.2 μl sY87-F, 0.2 μl sY87-R, 0.175 μl sY143-F, 0.175 μl sY143-R, 0.1 μl ex2DAZ-F, 0.1 μl ex2DAZ- R, 0.075 μl RhEY-F, 0.075 μl RhEY-R, 2.5 μl 10x buffer, 25 mM 1.5 μl MgCl 2 solution, mixed dNTPs solution 2μl, add dH 2 0 to 24 μl, in addition, 0.125 μl 250 U of Taq enzyme was added to each reaction system.

[0006] in:

[0007] The primer sequence for detection of sY87 gene deletion is:

[0008] Upstream: 5'-TAAAGCTCTGTTGCTTGAAAAGAGGG-3',

[0009] Downstream: 5'-TAGTGTGTAAATGGCACCATACATGACTA-3';

[0010] The primer sequences f...

specific Embodiment approach 2

[0019] Specific embodiment two: In this embodiment, PCR amplification is carried out to the Y chromosome fine deletion diagnostic reagent like this:

[0020] (1) Dissolve the dry powders of the following primers in TE with pH = 7-8: sY87-F, sY87-R, sY143-R, ex2DAZ-F, ex2DAZ-R, RhEY-F, RhEY-R, respectively to make 10 pmol / μl of sY87-F, sY87-R, sY143-R, ex2DAZ-F, ex2DAZ-R, RhEY-F, RhEY-R solutions;

[0021] (2) Add the following components to the 24 μl reaction system: 0.2 μl sY87-F, 0.2 μl sY87-R, 0.175 μl sY143-F, 0.175 μl sY143-R, 0.1 μl ex2DAZ-F, 0.1 μl ex2DAZ-R, 0.075 μl RhEY-F, 0.075 μl RhEY-R, 2.5 μl 10x buffer, 25 mM 1.5 μl MgCl 2 solution, mixed dNTPs solution 2μl, add dH 2 0 to 24 μl. In addition, 0.125 μl 250 U of Taq enzyme was added to each reaction system.

[0022] in:

[0023] The primer sequence for detection of sY87 gene deletion is:

[0024] Upstream: 5'-TAAAGCTCTGTTGCTTGAAAAGAGGG-3',

[0025] Downstream: 5'-TAGTGTGTAAATGGCACCATACATGACTA-3';

[0026] Th...

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Abstract

The invention relates to a reagent for diagnosing microdeletion of Y chromosome and a PCR amplification method therefor. The reagent is prepared by the following steps: (1) respectively dissolving the primer dry powders with TE whose pH value is between 7 and 8 to obtain a 10pmol / ª–l solution, (2) charging the following components into the 24ª–l reaction system: sY87-F, sY87-R, sY143-F, sY143-R, ex2DAZ-F, ex2DAZ-R, RhEY-F, RhEY-R, 10x buffer solution, MgCl2, dNTPs, Taq enzyme. The PCR amplification method comprises: charging a DNA sample into the reagent, pre-denaturing the reagent at 94íµ for 8 minutes, then denaturing respectively at 94íµ, 50íµ, 72íµ for 1 minute, at last cooling the reagent to 15íµ after 32 times circulations. The invention can provide a stable, sensitive and quick diagnosis, and the experimental cost is only one-tenth as much as the price of the overseas relative kits.

Description

Technical field: [0001] The invention relates to a reagent for diagnosing fine deletion of Y chromosome and a PCR amplification method for the reagent. Background technique: [0002] At present, 15% of couples of childbearing age suffer from infertility worldwide, and male infertility factors account for about 40%. An important reason affecting male infertility is genetic factors (gene and chromosomal abnormalities), among which Infertility due to genetic abnormalities accounts for 10-15% of male infertility. It has been reported in the literature that 90% of male infertility is caused by spermatogenesis dysfunction, of which idiopathic spermatogenesis disorder accounts for 60%, more than half of which are of unknown cause, and whether the Y chromosome is normal or not plays a crucial role. Therefore, it is of great clinical value to study the related genes of Y chromosome. In male patients with oligospermia and azoospermia, Y chromosome microdeletion accounts for 20.5%. D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68
Inventor 常东
Owner HARBIN MEDICAL UNIVERSITY
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