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高等学校化学学报

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利用荧光标记引物和DNA自动测序仪确定DNA的断裂位点

郑伟娟1, 陈媛1, 邵颖2, 唐忠华3, 郭子建2, 华子春1   

    1. 南京大学医药生物技术国家重点实验室,
    2. 南京大学配位化学国家重点实验室, 南京210093;
    3. 上海申能博彩生物科技有限公司, 上海200233
  • 收稿日期:2006-03-01 修回日期:1900-01-01 出版日期:2007-01-10 发布日期:2007-01-10
  • 通讯作者: 华子春

Determineation of DNA Cleavage Site Sequence Using Fluorescence Labeled Primer and DNA Sequencer

ZHENG Wei-Juan1, CHEN Yuan1, SHAO Ying2, TANG Zhong-Hua3, GUO Zi-Jian2, HUA Zi-Chun1   

    1. State Key Laboratory of Pharmaceutical Biotechnology,
    2. State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, China;
    3. Shanghai Shenergy Biocolor Bioscience ＆ Technology Company, Shanghai 200233, China
  • Received:2006-03-01 Revised:1900-01-01 Online:2007-01-10 Published:2007-01-10
  • Contact: HUA Zi-Chun

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被引次数

4

摘要： 建立了利用荧光标记引物和DNA自动测序仪进行DNA断裂位点分析的新方法, 该方法简便易行、灵敏度高、重复性好、数据分析客观性强、结果可靠, 适用于各种因素造成的DNA断裂位点的分析.

关键词: DNA切割, 断裂位点, 序列特异性, 荧光标记引物, DNA自动测序仪

Abstract: Determining the base sequence of DNA broken site is quite crucial for the study on the cleavage site specificity and mechanism of various natural or synthetic DNA cleavage regents, and on developing novel therapeutic drugs targeting at DNA. The most frequently used method depending on chemical reactions of the Maxam-Gilbert procedure, and the late arising methods used by Rui Ren et al. which were based on Sanger's DNA sequencing strategy, all had some deficiencies, either the pollution of radioactive materials, or really complicated and difficult to operate. In the present paper, a new method for DNA cleavage site sequence determination was developed. The fluorescence FAM-labeled primer was annealed to the DNA fragments, which has been cleaved by restriction enzymes or other regents, and extended along the template sequence. The products then loaded onto the polyacrylamide electrophoresis gel of ABI 377 DNA Sequencer. Data was collected and analyzed by using ABI PRISM Data Collection Software and ABI PRISM Sequencing Analysis Software. It is proved to be a credible and simple new approach to determine the base sequence of DNA broken sites.

Key words: DNA cleavage, Broken site, Sequence specificity, Fluorescence labeled primer, ABI 377 DNA Sequencer

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