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Analysis on the Gene component and Construction of the Fragment in 429P24 of China Merino MHC Class Ⅰ Section BAC Clone

Author: LiGang
Tutor: GaoJianFeng
School: Shihezi University
Course: Biochemistry and Molecular Biology
Keywords: labeled with 32P sheep MHC cDNA library probe
CLC: S826
Type: Master's thesis
Year: 2010
Downloads: 16
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Abstract


Objective:Study of sheep MHC class I genes composed of sections designed to find and research and an important economic traits, as well as with the sheep is closely related to the sense of resistance to disease-related genes. The 429P24BAC clone which contains genes in ovine MHC I region will be cut into fragments smaller than 2500bp by restriction enzyme BsaJ I and end labeled with [a-32P] dCTP. These radioactive label ed fragments can be used as probes to have hybridism with cDNA library. Positive cDNA clones will be marked by radioautography and we could get information and analysis of inserts in these positive clones by sequencing and data analysis in molecular biology.Methods:1. Pick up the China Merino Ovis aries’s MHC Class I 429P24 BAC clone from the BAC library,cultivanting and sending to the BeiJing HuaDa gene to sequence, to make a forecast about the sequence by eukaryote’s software as Genscan 1.0. analysis the forecast information. on NCBI to obtain the comment information.2. The 429P24 BAC clone which contains genes in ovine MHC I region is cut into fragments smaller than 2500bp by restriction enzyme BsaJ I and end labeled with [a-32P]dCTP in 3 ends.These radioactive labeled fragments are used as probes to have hybridism with cDNA library. Positive cDNA clones are marked by radioautography and we get information and analysis of inserts in these positive clones.Results:1. The result of the sequence information indicate that the length of this clone is 168kb.The forecast information results reveal that there are 13 genes on the clone. All of the 13 genes’ length is above 1000 bp. Blast this sequence on NCBI to attained the comment information.2.7 candidate positive cDNA clones were isolated using 429P24 BAC probes. Further analysis indicated these genes maybe related to immune and disease of sheep. The isolation of cDNA clones using BAC probes is effective and practical. The result revealed that these genes maybe related to immune and disease of sheep.3. Through the sequence obtained with the 429P24 BAC clone sequence alignment, results showed that the gene is indeed derived from Chinese Merino sheep MHC ClassⅠsection of the gene.Conclusion:1. To a large extent biological study is can not to break away from bioinformatics. Applying the bioinformatics methods can make our study better. When running into some complicated experiment question, especially about Genome sequence construction and functional study, bioinformatics is more important. Experiment results also indicated that using bioinformatics methods can more effective and shortcut to obtain the express genes. Furthermore this method is very simple and technique is very maturation.2. It is a feasibility methods of bacteriophage hybridization in situ in the hybridizate of library and library. Especially study some low express genes. This methods obtains the research worker’s favor for it’s high sensitivity, operation simple and short cycle. Experiment results also indicated that this method is feasibility.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Sheep
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