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
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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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