Location Of Dnase I Hypersensive Sites Exactly In The Promoter Region Of CD133 In Colonal Cancer Cell Line SW480 Cells By Inverse-pCR

Objective:Using the inverse PCR to locate DNase I enzyme-sensitive sites of gene CD133 promoter region in human colon cancer SW480 cells. And detect the available application of this method which could discover the genes regulate tumor cells.Methods:The human colon cancer cell line SW480 were selected for the study, and the cells were successfully cultured. Then 10 U / ml DNase I were used to cut and extract nucleus chromatin in SW480 cells. The method of phenol / chloroform / isoamyl alcohol(10: 10: 1)was used to extract the genomic DNA. And the restriction enzymes Eco RI and Xmal I had fragmented genomic DNA. The end of the DNA fragment was blunt, and then T4 ligase was used for PCR. And the product of inverse PCR amplification were purified and ligated into p MD-18 T carriers, then transferred into E.coli TOP10 for expression. Positive clones were sequenced.Results:By sequencing, 9 DNase I hypersensitivity sites which locate in upstream of the transcription initiation of CD133 gene in human colon cancer SW480 cells were identified.The DNase I hypersensitivity sites are located in the-700 ~-300 bp region of first exon.Conclusion:The inverse PCR technique can accurately determine the DNase I cleavage site of CD133 gene promoter region in human colon cancer SW480 cells, and these sites are gathered in a specific area.