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Primer for detecting separation purity of X, Y spermatozoon of cattle

A sperm and purity technology, applied in the field of nucleotide amplification, can solve the problems of long time, expensive fluorescent reagents, increased labor, etc., and achieve the effects of simple operation, reliable technical support, and low cost

Inactive Publication Date: 2008-06-11
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the FISH evaluation method requires the preparation of many samples in a short period of time, which virtually increases the amount of labor and takes a long time to complete the entire identification process. At the same time, the fluorescent reagents are expensive, which is not conducive to the popularization and application of this technology.

Method used

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  • Primer for detecting separation purity of X, Y spermatozoon of cattle

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1: Carry out PCR amplification with bovine blood DNA as template

[0020] The preparation of bovine genomic DNA by alkaline lysis method requires two parts: alkaline lysis solution and neutralization solution. The components of alkaline lysis solution include: KOH 500mmol / L, DTT 125mmol / L; the components of neutralization solution include: Tris.HCl 900mmol / L, pH=8.3.

[0021] Using a bull and a cow as materials, take 10 μL of anticoagulated blood each into a 0.2 mL high-pressure sterilized PCR tube, add 90 μl of double-distilled water, and mix well. Centrifuge at 12000rpm for 2-3 minutes, discard the supernatant liquid. Add 10 μl of alkaline lysate and mix well by pipetting, lyse at 65°C for 10 minutes, add 25 μl of neutralizing solution, pipette and mix well for PCR detection, or store at -20°C for later use.

[0022] Primer design

[0023] According to the sequence of Y chromosome sex-determining gene Sry (gi: 4878004, the sequence report length is 2751...

Embodiment 2

[0040] Embodiment 2: single sperm PCR amplification

[0041] 1. Separation of single sperm by agarose spread

[0042] 1.1 Wash sperm with sucrose solution (250mM sucrose, 5mM Tris. base)

[0043] a. Take an autoclaved 1.5ml centrifuge tube.

[0044] b. Take out a tube of frozen semen from liquid nitrogen and put it into 40℃ water to thaw instantly.

[0045] c. Cut off one end of the thin tube cotton plug with scissors, align the mouth of the cut cotton plug with the mouth of a 1.5ml centrifuge tube, cut off the other end with scissors, the sperm flow into the 1.5ml centrifuge tube along the mouth of the tube, and pipette the remaining sperm with 10μl blown into the centrifuge tube.

[0046] d. Add 5 μl of undiluted sperm into 495 μl of deionized water, and mix well with a pipette.

[0047] e. Take about 20 μl of mixed sperm and cover the entire cell counting plate, and count the number of sperm under a 10-fold ordinary optical microscope.

[0048] f. Centrifuge the undilute...

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Abstract

The invention provides a primer for detecting the X and Y sperm separation purity. Aiming at a sex-determining gene Sry on a bull Y chromosome, the primer is designed through the PCR mismatching technology. The fragment size can be amplified by 295 bp through the primer, in order to prevent false positive from appearing, a pair of internal control primers C34 is designed through the invention according to a bull autosome 3 reported sequence, the fragment size is amplified by 208bp, dual PCR amplification is performed to single bull sperm through the two pairs of primers, and then the final evaluation is performed to the sperm separation purity according to the statistical analysis to the detection result. The invention provides the technology used for identifying the bull X and Y sperm separation purity with low cost and simple, rapid, and accurate operation, and provides reliable technical support for the popularization and the application of the subsequent sexing semen and the optimization of a sperm separation method.

Description

technical field [0001] The invention relates to nucleotide amplification technology, in particular to a primer for detecting the separation purity of bovine X and Y sperm, the primer can identify the purity of the separated bovine X and Y sperm, so as to play a sex control role in the process of bovine reproduction role. Background technique [0002] Gender control has great application value in production practice. With the help of gender control technology, livestock of different genders can be selected to meet production needs. X and Y sperm separation is the most fundamental technical means of gender control. At the same time, the accuracy of X and Y sperm separation The degree is directly related to the effect of gender control, so the establishment of a fast and accurate evaluation system is conducive to the optimization and improvement of the separation method. In addition, the simple and quick evaluation method is directly related to the credibility of sex control, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C07H21/04
Inventor 王栋朱化彬郭家明程金华郝海生杜卫华张林波
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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