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Specific primers and typing method of class ii mhc gene for detection of antibacterial potential of crested ibis
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A crested ibis and primer pair technology, applied in the field of genotyping, can solve the problems of serious inbreeding, high mortality and disability rate of young birds, and low reproductive ability.
Active Publication Date: 2017-03-22
ZHEJIANG UNIV
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After decades of protection work, although the population of crested ibises is gradually recovering, it is still not out of the endangered situation. Since the known individuals in the world are all descendants of two pairs of crested ibises rediscovered in 1981, inbreeding is serious. There are problems such as low reproductive ability and high rate of death and disability of juvenile birds
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example 1
[0043] 1. Sample preparation: Crested ibis blood and tissue samples came from artificial populations such as Deqing, Sado, Beijing, Henan, Yangxian and Louguantai.
[0044] 2. DNA extraction: phenol chloroform method to extract blood and tissue DNA.
[0045] 3. Primer synthesis: According to the obtained crested ibis class II-MHC gene sequence, specific primers for the II-A1 site were designed to amplify exon 2 for genotyping of the modified site, and BGI synthesized primers. The primer sequences are as follows:
[0046]
[0047] 4. Primer amplification: apply the above amplification primers to PCR respectively to amplify the extracted sample DNA. Set the PCR thermal cycle annealing temperature range to 64°C.
[0048] 5. Amplification result detection: take 2 ul of the above-mentioned PCR amplification products and carry out agarose gel electrophoresis, the gel concentration is 1.5%. The result is as figure 1 As shown, this primer can effectively amplify the target band, ...
example 2
[0062] 1. Sample preparation: Crested ibis blood and tissue samples came from artificial populations such as Deqing, Sado, Beijing, Henan, Yangxian and Louguantai.
[0063] 2. DNA extraction: phenol chloroform method to extract blood and tissue DNA.
[0064] 3. Primer synthesis: According to the obtained crested ibis class II MHC gene sequence, specific primers for the II-B1 site were designed to amplify exon 2, and the primers were synthesized by BGI. The primer sequences are as follows:
[0065]
[0066] 4. Primer amplification: apply the above amplification primers to PCR respectively to amplify the extracted sample DNA. Set the PCR thermal cycle annealing temperature range to 63°C.
[0067] 5. Amplification result detection: take 2 ul of the above-mentioned PCR amplification products and carry out agarose gel electrophoresis, the gel concentration is 1.5%. The result is as figure 2 As shown, this primer can effectively amplify the target band, the fragment size of ...
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Abstract
The invention discloses a specific primer and a gene parting method for an II-type MHC gene for anti-bacterial potential detection on crested ibis. The sequence of the specific primer is as shown in SEQ ID NO.1-SEQ ID NO.7. By adopting the amplification primer disclosed by the invention, nucleotide sequences of different sites of II-type MHC of crested ibis can be effectively amplified, and each body can be subjected to gene parting by using a later SSCP-HD technique. The specific primer has the advantages of high specificity, simplicity and convenience and rapidness in operation; due to gene parting on the II-type MHC gene, polymorphism of the II-type MHC gene of crested ibis can be evaluated, the potential of bacterial disease resistance of crested ibis can be detected, and important reference for working such as new population founder selection, individual wild running and artificial pairing in crested ibis protection is provided.
Description
technical field [0001] The invention provides primers for amplifying the gene sequence of each site of MHC class II of the crested ibis and a genotyping method based on the single-strand conformation polymorphism (Single-Strand Conformation Polymor-phism, SSCP) technology. Background technique [0002] Crested ibis, an endangered species in the world and a national and key protected animal, belongs to the genus Crested Ibis of the Ibis family of the order Aves. It is a medium-sized wading bird that was once widely distributed in China, Russia, Japan and the Korean Peninsula in East Asia. At the end of the 19th century and the beginning of the 20th century, due to human activities and other reasons, the population of crested ibises decreased significantly, and wild individuals in Russia, the Korean Peninsula and Japan became extinct one after another. Until 1981, researchers rediscovered 7 wild crested ibises in Qinling Mountains, Yang County, Shaanxi Province, my country. The...
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