Location of DNase Ⅰ hypersensive sites exactly in the promoter region of CD133 in colonel cancer cell line SW480 cells by inverse-PCR / 肿瘤研究与临床

ABSTRACT

Objective To precious localize DNase Ⅰ hypersensive sites exactly in the promoter region of CD133 of cell line SW480 by inverse-PCR.Methods The colonel cancer cell SW480 nuclei were suspended in digested buffer,treated with DNase Ⅰ at the concentration of 10 U/ml for 10 min.The inversePCR was performed as follows.DNA treated by DNase Ⅰ was purified,fragmented with restricted enzyme EcoRI and Xmal Ⅰ.Then the ends were blunted,ligated by T4 ligase.PCR was performed,and production was sequenced.The restricted enzymes cut sites were near DNase Ⅰ cleavage sites.Results 9 DNase Ⅰ cut sites were identified in CD133 promoter region.The DNaseI hypersensitive sites all distributed in a region -300 bp--700 bp up to transcription start site.Conclusion The DNase Ⅰ cleavage sites could identified preciously by application of inverse-PCR.These sites locate in a region of-300 bp--700 bp up to transcription start site.