| CHEN Xiao-sui,FENG Jiang-bin,ZHOU Li-jun,et al.Construction and confirmation of the plasmid of human mitochondrial DNA 4977 bp deletion induced by ionizing radiation[J].Chinese Journal of Radiological Medicine and Protection,2006,26(1):52-54 |
| Construction and confirmation of the plasmid of human mitochondrial DNA 4977 bp deletion induced by ionizing radiation |
| Received:April 24, 2005 |
| DOI： |
| KeyWords:Ionizing radiation Mitochondrial 4977 bp deletion Plasmid construction |
| FundProject:北京市自然科学基金资助项目(7053073) |
| Author Name | Affiliation | E-mail | | CHEN Xiao-sui | Chinese Naval General Hospital,Beijing 100037,China | qjliu@nirp.cn | | FENG Jiang-bin | Chinese Naval General Hospital,Beijing 100037,China | | | ZHOU Li-jun | 100037 北京,海军总医院 | | | 陆雪 | Chinese Naval General Hospital,Beijing 100037,China | | | 王欲晓 | Chinese Naval General Hospital,Beijing 100037,China | | | 陈德清 | Chinese Naval General Hospital,Beijing 100037,China | | | 曲佳 | 100037 北京,海军总医院 | | | 刘青杰 | Chinese Naval General Hospital,Beijing 100037,China | |
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| Abstract:: |
| 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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