| 陈晓穗,封江彬,周丽君,陆雪,王欲晓,陈德清,曲佳,刘青杰.电离辐射致线粒体DNA 4977 bp缺失质粒构建及其鉴定[J].中华放射医学与防护杂志,2006,26(1):52-54 |
| 电离辐射致线粒体DNA 4977 bp缺失质粒构建及其鉴定 |
| Construction and confirmation of the plasmid of human mitochondrial DNA 4977 bp deletion induced by ionizing radiation |
| 投稿时间：2005-04-24 |
| DOI： |
| 中文关键词: 电离辐射 线粒体DNA4977bp缺失 质粒构建 |
| 英文关键词:Ionizing radiation Mitochondrial 4977 bp deletion Plasmid construction |
| 基金项目:北京市自然科学基金资助项目(7053073) |
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| 摘要点击次数: 2893 |
| 全文下载次数: 1999 |
| 中文摘要: |
| 目的 建立电离辐射诱导的人线粒体DNA(mtDNA)4977bp缺失片段和对照DNA片段的稳定质粒,用于线粒体基因组相关研究。方法 对无mtDNA4977bp缺失的正常人外周血进行离体照射10Gy60Coγ射线,提取细胞总DNA,用巢式PCR扩增mtDNA4977bp缺失片段,普通PCR扩增对照ND1基因片段。PCR产物经纯化后构建质粒;提取质粒DNA,DNA样品经酶切、纯化后测序,将测序结果 进行BLAST分析。结果 PCR扩增的跨mtDNA4977bp缺失的DNA片段和ND1基因片段大小与预期值是吻合的。对制备的质粒DNA样品进行测序,结果 经BLAST分析,两种质粒DNA与人线粒体序列同源性在99%以上。结论 本研究中所构建的质粒是成功的,可以用于人mtDNA4977bp缺失的定性或定量研究。 |
| 英文摘要: |
| Objective To construct a stable plasmid that spanning deleted human mitochondrial DNA(mtDNA) 4977 bp induced by ionizing radiation and another one for control DNA fragment,in order to use in the human mitochondrial genome study in the future.Methods The peripheral blood,which had no mtDNA 4977 bp deletion found in previous study,was exposed to 10 Gy 60Co γ-rays in vitro.The total cell DNA was extracted and PCR was carried out: a nest-PCR of three-round PCR was used for the mtDNA 4977 bp deletion and one-round regular PCR was used for the control ND1 gene.The PCR products were used for transfection by electroporation and the positive clones were obtained after screening.The plasmid DNA was isolated and sequenced after enzymatic digestion and purification.The sequence result was BLASTed with the human mitochondrial genome.Results The sizes of PCR products for the flanked 4977 bp deletion and the ND1 gene were similar with those predicted according to GeneBank.The sequences for the positive clones were above 99 per cent homologous with the human mitochondrial genome after BLASTed.Conclusion The plasmids for deleted human mtDNA 4977 bp and control DNA fragment have been constructed successfully,and they could be used in the quality and quantity studies on human mtDNA 4977 bp deletion. |
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