CAJ | 학술논문

目的利用反向PCR法精确定位人结肠癌SW480细胞CD133基因启动子区对脱氧核糖核酸酶Ⅰ(DNase Ⅰ)酶切敏感的位点.方法提取SW480细胞的细胞核,以10 U/ml DNase Ⅰ处理细胞核10 min.反向PCR扩增:提取基因组,用限制性内切酶EcoR Ⅰ和Xmal Ⅰ片段化基因组,末端补平,T4连接酶连接,利用反向PCR后对产物测序,靠近限制性内切酶位点的序列即DNase Ⅰ的切割位点.结果 CD133基因转录起始点上游9个DNase Ⅰ高敏感位被鉴别出,它们位于第一个外显子-700 ～-300 bp碱基区域内.结论用反向成环PCR技术可以精确测定DNase Ⅰ的剪切位点,这些位点聚集在一个特定的区域内.
목적 이용반향PCR법정학정위인결장암SW480세포CD133기인계동자구대탈양핵당핵산매Ⅰ(DNase Ⅰ)매절민감적위점.방법 제취SW480세포적세포핵,이10 U/ml DNase Ⅰ처리세포핵10 min.반향PCR확증:제취기인조,용한제성내절매EcoR Ⅰ화Xmal Ⅰ편단화기인조,말단보평,T4련접매련접,이용반향PCR후대산물측서,고근한제성내절매위점적서렬즉DNase Ⅰ적절할위점.결과 CD133기인전록기시점상유9개DNase Ⅰ고민감위피감별출,타문위우제일개외현자-700 ～-300 bp감기구역내.결론 용반향성배PCR기술가이정학측정DNase Ⅰ적전절위점,저사위점취집재일개특정적구역내.
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.