Location of DNaseI hypersensive sites exactly in the promoter region of CD133 in colonel cancer cell line SW480 cells by inverse-PCR

Abstract

Objective To precious localize DNaseⅠ 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 inverse-PCR 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. Key words: Colonic neoplasms; Inverse-PCR; Deoxyribonuclease Ⅰ; Transcription initiation site