Specific primer sequence of cattle Y-chromosome and its application in cow embryo six identification

A primer sequence and chromosome technology, applied in the field of bovine Y-chromosome-specific primer sequences, can solve the problems of affecting the accuracy of embryo sex identification, affecting identification results, limited sensitivity, etc., and achieve improved sensitivity and specificity, low homology. , the effect of avoiding pollution

Inactive Publication Date: 2002-12-11
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the sequences commonly used to identify the sex of bovine embryos include Y-chromosomal repeat sequences and SRY gene core sequences, etc., but Y-chromosome repeat sequences often affect the accuracy of embryo sex identification because of the possible presence of homologous sequences on other chromosomes The SRY core sequence used by Zeng Yitao and others also has its shortcomings, because the core sequence of the SRY gene has a high homology (up to more than 80%) among mammals, such as the homology with the core sequence of the SRY gene of people. As high as 84%, the DNA of male operators and other animals is likely to contaminate the identification process when carrying out sex identification of bovine embryos, especially when performing PCR operations in the field, because the environmental conditions are not strict enough, it is easy to produce false positives, thus Affects the accuracy of embryo sex identification
In addition, the sensitivity of one-time amplification of conventional PCR used by Herr CM and others is limited, because the number of cells that can be cut and sampled from the embryo is very small during embryo sex identification, so sometimes the amount of amplification product is too small to affect the judgment of the identification result

Method used

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  • Specific primer sequence of cattle Y-chromosome and its application in cow embryo six identification
  • Specific primer sequence of cattle Y-chromosome and its application in cow embryo six identification
  • Specific primer sequence of cattle Y-chromosome and its application in cow embryo six identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Carry out gender identification through bovine genomic DNA, and detect the specificity of primers.

[0023] Ear tissue DNA of bulls and cows was extracted first, then the extracted bovine ear tissue DNA was dissolved in 400 μl sterilized ultrapure water, and then 0.5 μl was used for nested PCR.

[0024] According to the SRY gene 5' regulatory region sequence in the Y-chromosome-specific DNA sequence provided by Genbank, the primer sequence designed and synthesized by the present invention is as follows:

[0025] Primer sequence

[0026] A 5′-AGCATTCTGAAAGTCGTTAGCAC-3′

[0027] B 5′-ATGAAGGTGCAGCAGACATACTA-3′

[0028] C 5′-AGCTACTGGAATACGTACATAGA-3′

[0029] The specific experimental process of using the primers designed and synthesized by the present invention for gender identification is as follows:

[0030] The reaction system of nested PCR is as follows: the volume of the first PCR reaction system is 10 μl, which contains 0.1 μmol / l each of prime...

Embodiment 2

[0032] Example 2: Bovine Embryo Sex Identification

[0033] Extraction of trace bovine embryonic cell DNA: Use a simple embryo divider to cut 4-6 cells from morula or blastocyst, then put the embryonic cell sample into a 200μl centrifuge tube, add 7μl sterilized ultrapure water; put it on the centrifuge Centrifuge briefly to make the embryonic cell samples exist at the bottom of the centrifuge tube, seal the centrifuge tube with a sealing film; immediately place it on ice after boiling at 100°C for 10-15min; after cooling, centrifuge at 14000r / min for 10min, and take the supernatant for PCR. The reaction system and cycle parameters of the nested PCR are the same as in Example 1. The sex identification results of the embryos are attached figure 2 shown. Male embryos can amplify a 205bp Y-chromosome-specific fragment and a 403bp casein gene fragment, while female embryos can only amplify a 403bp casein gene fragment. The sex of 12 embryos was detected, and the sex of calving...

Embodiment 3

[0034]Example 3: Use the primers designed in the present invention to perform PCR amplification on human, sheep and pig genomic DNA, and check whether the primers are bovine specific.

[0035] The blood of humans, sheep and pigs was collected, and after DNA was extracted, it was used for nested PCR amplification. The specific experimental process was the same as in Example 1. As a result, none of the genomic DNAs of human, sheep and pigs had any amplified products, indicating that the primers designed by the present invention are strictly cattle-specific, and DNA from male operators and other animals will not pollute the results of sex identification of bovine embryos.

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Abstract

The present invention discloses a specific primer sequence of cattle Y-chromosome and its application in cattle embryo six identification. Cattle ear tissue DNA is first extracted to perform primer specificity identification; and embryo cell sample DNA is then extracted to perform PCR sex identification. The bull sample may be amplified to 255 bp segment when external primer is used in PCR reaction and to 205 bp when internal primer is used, while cow sample has no any amplified product, and this shows the bull specificity of the primer and the applicability of the primer in identifying cattle embryo sex. The present invnetion has the advantages of less pollution, high accuracy and high sensitivity and is suitable for field operation.

Description

technical field [0001] The invention relates to a bovine Y-chromosome-specific primer sequence for identifying the sex of bovine early embryos by means of nested PCR. Background technique [0002] In the field of agricultural biotechnology, sex control, especially the sex control of large livestock such as cattle, has long been one of the major theoretical topics that biological scientists have devoted themselves to research, and has huge economic value. There are two main ways to realize the artificial control of the sex of domestic animals: one is the separation of X sperm and Y sperm; the other is the sex identification of early embryos. However, judging from the current X and Y sperm separation technology, although the use of flow cytometry has successfully separated X and Y sperm, the cost is high and the separation efficiency is low, so it is difficult to promote it in practical applications for a while. With the industrialization of embryo transfer technology, the se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12Q1/68
Inventor 黄路生陈从英陈静波任军陈克飞丁能水高军艾华水
Owner JIANGXI AGRICULTURAL UNIVERSITY
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