Beef embryo sex simple identification method

An identification method, the technology of bovine embryos, applied in the field of agricultural biology, can solve the problems of identification reaction results, cumbersome and time-consuming operations, etc., and achieve the effect of simple technology, high sensitivity, and improved identification efficiency

Inactive Publication Date: 2011-05-25
西北农林科技大学生物工程研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the operation is cumbersome and time-consuming, and requires expensive equipment such as PCR instrument, electrophoresis instrument and gel imaging system
[0003] At present, there is no report on the use of dNTP precipitation method to identify the reaction results at home and abroad.

Method used

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  • Beef embryo sex simple identification method
  • Beef embryo sex simple identification method
  • Beef embryo sex simple identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] The blood of male and female cows was collected, and 20% ACD blood anticoagulant was added thereto, and genomic DNA was extracted using a whole blood genome extraction kit (Takara), and diluted to 5 ng / μL for use.

[0148] This test uses bovine-specific primers and bovine-male-specific primers, and the primer sequences are as follows:

[0149] B3P1 TTGTGTCCAGAAGCCAATGT

[0150] B3P2 AGGAGGCTTGACTCCCTT

[0151] B3P3 AGATCCCTGTCGCCCCT

[0152] B3P4 GGGTATCTCTTGGAGCTCACTG

[0153] B3P5 TGCGTCTGGAATGCAACCCCTTGTGTCCAGAAGCCAATGT

[0154] B3P6 CAAAGAACCCCGCTCTGCTCTAGGAGGCTTGACTCCCTT

[0155] S4P1CCTGTCCGACTCTTTGCG

[0156] S4P2 GTGGCTCAGAAGGTAAAGTGTC

[0157] S4P3 ATGGATGGAGGAGCCTGATAG

[0158] S4P4 GAATCAAACCCAGGTCTCC

[0159] S4P5 TGACAATCCACCCCAGCACTCCCTGTCCGACTCTTTGCG

[0160] S4P6 CTCCAGGGGATCTTCCTGACCGTGGCTCAGAAGGTAAAGTGTC

[0161] The reaction volume was 25 μL, including 1× buffer (20 mM Tris-HCl, pH 8.8; 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1%...

Embodiment 2

[0166] This test was carried out at the production base (Keyuan Company, Yangling Demonstration Zone). Fresh bovine embryos, including blastocysts and morula embryos (A grade), were cut using a micromanipulator. The cut was about 10%, and then the method was used to make Embryos are sexed and transferred to recipient cattle.

[0167] Use primers as:

[0168] P1 CCTGTCCGACTCTTTGCG

[0169] P2 ACATTTTGCTAAGGGCTTCC

[0170] P3 ATCCCATGGATGGAGGAGC

[0171] P4 CAGGAATCAAAACCCAGGTCTCC

[0172] P5 CCAGCACTCTTGCCTGGAAACCATGGACTGTCGCCTAT

[0173] P6 TTCCTTCTCCAGGGGATCTTCCAGAAGGTAAAGTGTCTGCCT

[0174] P1 AGGGCCAATAGACCTCATCT

[0175] P2 TCGCGACCTCTCTCCAAAC

[0176] P3 AGATCCCTGTCGCCCCT

[0177] P4 GGGTATCTCTTGGAGCTCACTG

[0178] P5 TGCGTCTGGAATGCAACCCCTTGTGTCCAGAAGCCAATGT

[0179] P6 CAAAGAACCCCGCTCTGCTCTAGGAGGCTTGACTCCCTT

[0180] Add the operating solution (5 μL) containing embryonic cells into a transparent PCR tube, and then add 19 μL of solution A in the kit, including 1...

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Abstract

The present invention relates to a convenient sex determination method of bovine embryos, the identification rate of the blood genome achieves 100%, and the identification rate of the embryo sex exceeds 99.0%. This method has convenient operation, high sensitivity, good practicability and simple equipment, and can determine the embryo sex in 1 hour. The formed detection kit of embryo sex can be applied to production absolutely and the cost is only 1 / 10 of products of the same kind. In the present invention 6 specific primers are designed according to the bovine specific sequences and male specific sequences, and 6 specific sites can be recognized. Large amount of DNA fragments of different sizes are amplified in a short time using a dumbbell-shaped structure unit as a core that is formed by the target sequences. The only requirement is aqueous thermostat at 60-65DEG C. dNTPs precipitation is used in the determination of the result (The negative result is that a white band appears in the middle part of the reaction tube).

Description

1. Technical field: [0001] The invention relates to a method for identifying the sex of an embryo in the field of agricultural biotechnology, in particular to a simple method for identifying the sex of a bovine embryo. 2. Background technology: [0002] With the continuous improvement of animal reproduction technology, the dairy cow embryo transfer industry has developed rapidly, such as estrus synchronization, superovulation, embryo transfer and other technologies are becoming mature, providing technical support for the rapid breeding of fine-bred dairy cows. In the breeding process of dairy cows, the sex of calving has a huge impact on economic benefits, and the production of cows has become a technical difficulty that needs to be overcome urgently in this field. At present, the production of sex-controlled embryos mainly includes embryo sex identification and sex-controlled semen. These two methods have their own advantages and disadvantages. Traditional embryo sex identi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 张志平张涌刘俊平安志兴张君涛张立权富生郑月茂赛务加甫蔡欣
Owner 西北农林科技大学生物工程研究所
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