Method for detecting separation purity of bull sperms X and Y

A sperm and purity technology, applied in the field of molecular biology, can solve the problems of expensive fluorescent reagents, unfavorable popularization and application, and increased labor load, and achieve the effects of fast and sensitive technical support, avoiding technical operations, and reducing detection costs

Inactive Publication Date: 2011-01-05
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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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 com

Method used

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  • Method for detecting separation purity of bull sperms X and Y
  • Method for detecting separation purity of bull sperms X and Y
  • Method for detecting separation purity of bull sperms X and Y

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1: Carry out PCR amplification with bovine blood DNA and semen DNA as template

[0029] 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.

[0030] Take 10 μL of anticoagulated blood from bull and cow into 0.2 mL high-pressure sterilized PCR tubes, add 90 μL of double distilled water, and mix well. Centrifuge at 12000rpm for 2-3 minutes, discard the upper liquid; for bull semen, take a semen and use 1ml of 0.25% sucrose solution, pipette and mix, and centrifuge at 12000rpm for 10 minutes, which can be repeated twice. Add 10 μl of alkaline lysate to blood cells or sperm cells and mix well by pipetting, lyse at 65°C for 10 minutes, add 25 μl neutralizing solution, pipette and mix well, and use immediately for PCR detec...

Embodiment 2

[0042] Embodiment 2: single sperm PCR amplification

[0043] 1. Separation of single sperm by agarose spread

[0044] 1.1 Wash sperm with sucrose solution (250mM sucrose, 5mM Tris. base)

[0045] a. Take 3 autoclaved 1.5ml centrifuge tubes.

[0046] b. Take out one regular semen, X semen, and Y semen from the liquid nitrogen, and put them in 40°C water to thaw instantly.

[0047] 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.

[0048] d. Add 5 μl of undiluted sperm into 495 μl of deionized water, use a pipette to mix well, take about 20 μl of mixed sperm to cover the entire cell counting plate, and count the number of sperm under an ordinary optical microscope at 10 times.

[0049] e. Centrifug...

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Abstract

The invention relates to a primer for detecting the sex of a single bull sperm or the purities of sperms X and Y in bull separation semen and application thereof. The primer comprises nucleotide sequences shown as SEQ ID NO.1 and 2. A method comprises the following steps of: performing amplification detection on the primer by a rapid polymerase chain reaction (PCR) method and identifying the sex of a single sperm through an agarose gel electrophoresis result according to a difference between the lengths of specific amplification products of two chromosomes; and performing single PCR amplification and agarose gel electrophoresis on each single bull sperm separated from the separation semen and performing statistical analysis on a single sperm sex detection result so as to make a final evaluation on the purity of the separation sperm. A specific PCR product can be amplified in any sperm cell by the method without designing any internal standard primer, the method is technology having the advantages of low cost, simple and convenient operation and rapid and correct identification of the separation purity of bull sperms X and Y and reliable and necessary technical support is provided for the popularization of subsequent control of semen and optimization and innovation of a sperm separation method.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting the separation purity of bovine X and Y sperm. Background technique [0002] Gender control has great application value in animal husbandry production. 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 sex control, so the establishment of a fast and accurate evaluation system is conducive to the optimization and improvement of separation methods, and it is also related to the credibility of sex control technology, which is helpful for the promotion and market popularization of sex-controlled semen. [0003] At present, the evaluation method of flow cytometry reanalysis and the evaluation method of fluore...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 王栋朱化彬林博程金华吴承疆郝海生杜卫华赵学明
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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