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Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof

A detection kit and Y chromosome technology, applied in the field of cytomolecular genetics, can solve the problems of increased economic expenditure, laborious operation, expensive DNA extraction reagents, etc., and achieve the goal of less blood consumption, less pain, and reduced economic expenditure Effect

Inactive Publication Date: 2015-06-03
NINGBO FIRST HOSPITAL
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

(2) The operation is laborious and laborious
When the sample size is large, extracting DNA will take up a considerable amount of time and energy for the experimenter
(3) Increase economic expenditure, the more efficient the DNA extraction reagent is, the more expensive it is
The cost of extracting DNA is even much higher than the PCR reaction itself
(4) Even with DNA extraction, in a small number of special samples, it is difficult to remove PCR reaction inhibitors
[0006] Chinese patent 200710074317.X discloses male Y chromosome microdeletion detection kit and detection method, and 200910094618.8 discloses a gene detection method for Y chromosome microdeletion, both of which adopt the method of extracting DNA from peripheral blood and using multiplex PCR amplification , the operation is time-consuming and labor-intensive, increasing economic expenditure

Method used

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  • Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof
  • Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A method for rapid gene detection of Y chromosome microdeletion, the specific method is as follows:

[0058] 1. Peripheral blood collection: use a heparin anticoagulant tube to extract 1ml of peripheral blood and mix well for later use;

[0059] 2. Multiplex PCR system amplification Multiplex PCR is divided into two sets of systems, Group A and Group B.

[0060] Group A PCR reaction system: The PCR reaction system of 25 μl is: PCR buffer buffer: 300mM Ttricine, 25mM (NH 4 ) 2 SO 4 , 17.5 mM MgCl 2 , 30% glycerol, 5 μl; 1X mixed primer A 2.5 μl; whole blood 2.5 μl; 2 μl 2.5mM dNTPs (Takara); 1 μl 100 units / μl mutant Tag enzyme (New England Biotechnology (Beijing) Co., Ltd.); ddH 2 O 12 μl.

[0061] Preparation of Group A 5X mixed primers (mixed primers A): the content of each synthetic primer is 1OD, ZFY-F 3.7nmol / OD, ZFY-R 3.6nmol / OD, SRY-F 4.8nmol / OD, SRY-R 4.9nmol / OD, SY254-F 4.2nmol / OD, SY254-R 4.1nmol / OD, SY86-F 4.3nmol / OD, SY86-R 4.3nmol / OD, SY127-F 3.9nmol / O...

Embodiment 2

[0110] A kit for rapid detection of male Y chromosome microdeletions, comprising: PCR reaction buffer, dNTP, DNA polymerase, mixed multiple amplification composition A liquid and B liquid.

[0111] PCR buffer includes 300mM Ttricine, 25mM (NH 4 ) 2 SO 4 , 17.5 mM MgCl 2 , 30% Glycerin.

[0112] Preparation of 5X amplification composition A solution: the content of each tube of all synthetic primers is 1OD, ZFY-F 3.7nmol / OD, ZFY-R 3.6nmol / OD, SRY-F 4.8nmol / OD, SRY-R 4.9nmol / OD , SY254-F 4.2nmol / OD, SY254-R 4.1nmol / OD, SY86-F 4.3nmol / OD, SY86-R 4.3nmol / OD, SY127-F 3.9nmol / OD, SY127-R 4.2nmol / OD. Take 1 tube of ZFY-F and add 250μl deionized water, mix well, transfer all the liquid to ZFY-R tube, mix well, repeat the above steps, transfer SRY-F, SRY-R, SY254-F, SY254-R, SY86-F, SY86-R, SY127-F and SY127-R were all dissolved in 250 μl deionized water, aliquoted, and the primer information was indicated.

[0113] The sequence of each primer in solution A:

[0114] Internal co...

Embodiment 3

[0146] Example 3 Method for detecting male Y chromosome microdeletion using this kit

[0147] 1. Peripheral blood collection: use a heparin anticoagulant tube to extract 1ml of peripheral blood and mix well for later use;

[0148] 2. Multiplex PCR system amplification

[0149] Multiplex PCR is divided into two systems, group A and group B

[0150]10. Group A PCR reaction system: 25 μl of PCR reaction system includes: PCR reaction buffer: 300mM Ttricine, 25mM (NH 4 ) 2 SO 4 , 17.5 mM MgCl 2 , 30% glycerol, 5 μl; whole blood 2.5 μl; 2 μl 2.5mM dNTPs; 1 μl 100 units / μl mutant Tag enzyme Hemo Klen Tag (New England Biotechnology (Beijing) Co., Ltd.); 1.25-2.5 μM mixed combination primer 2.5 μl ; 12 μl H 2 O.

[0151] Group B PCR reaction system: the same as tube A reaction system, in which the primers are: 1X mixed primer B 2.5 μl.

[0152] The two sets of systems in Group A and Group B were subjected to thermal cycle reactions at the following temperatures: initial pre-den...

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Abstract

The invention belongs to the field of molecular cytogenetics and relates to an amplified composition for fast gene detection of microdeletion of Y chromosomes, a kit containing the same and an application thereof. The amplified composition comprises a group A and a group B, wherein each group comprises a pair of primers respectively in AZFa, AZFb and AZFc areas and two pairs of internal control primers ZFY and SRY; the primers in the group A have the nucleotide sequence shown in SEQ ID No.1-1O; the primers in the group B have the nucleotide sequence shown in SEQ ID No.11-2O. The amplified composition, the kit and the application have the advantages that on the basis of the prior art, multiple PCR (Polymerase Chain Reaction) buffering liquid and Tag polymerase are improved, and trace whole-blood amplification is directly adopted due to less blood consumption, so that the pain of a patient is reduced; and the procedure of extracting DNA is omitted, so that the time and the labor are saved and the economic expenditure is greatly reduced.

Description

technical field [0001] The invention belongs to the field of cytomolecular genetics, and relates to an amplification composition for rapid gene detection of Y chromosome microdeletion, a kit containing the amplification composition and an application thereof. Background technique [0002] Y chromosome microdeletion is an important reason for male infertility or severe oligozoospermia. Among patients with male infertility caused by spermatogenesis disorders, the incidence of Y chromosome microdeletion is the second genetic factor, second only to Klinefelter syndrome. Based on the importance of the detection of Y chromosome microdeletions, the European Society of Andrology and the European Molecular Genetics Quality Network jointly launched the guidelines for the detection of Y chromosome microdeletions, making it a standardized inspection item. At present, andrology requires Y chromosome microdeletion gene detection as a routine examination of male infertility. On the one ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 马琪程跃陈俊丰马超虞碧霞
Owner NINGBO FIRST HOSPITAL
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