生物技术通报 ›› 2016, Vol. 32 ›› Issue (4): 210-216.doi: 10.13560/j.cnki.biotech.bull.1985.2016.04.028

• 研究报告 • 上一篇    下一篇

稳态和时间分辨荧光光谱法检测pBR322质粒中十字形结构

夏岚12,张旭23   

  1. 1. 南京工业大学生物与制药工程学院,南京 211816;
    2. 盐城师范学院药学院,盐城 224051;
    3. 南京大学医学院,南京 210093
  • 收稿日期:2016-02-25 出版日期:2016-04-25 发布日期:2016-04-26
  • 作者简介:夏岚,女,硕士研究生,研究方向:抗瘤大分子的活性研究;E-mail:XLxialan@163.com
  • 基金资助:
    江苏省六大人才高峰资助项目(swyy-030)

Steady-state and Time-resolved Fluorescence Spectroscopy to Detect the Cruciform Structure in pBR322

XIA Lan12 ,ZHANG Xu23   

  1. 1. College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211816;
    2. School of Pharmacy,Yancheng Teachers University,Yancheng 224051;
    3. Medical School of Nanjing University,Nanjing 210093
  • Received:2016-02-25 Published:2016-04-25 Online:2016-04-26

摘要: 在目的DNA不被酶切成不同片段的情况下,应用光谱方法探测pBR322质粒中十字形结构DNA的形成。吡咯脱氧胞苷(Pyrrolo deoxycytidine,PdC)取代dC掺入到pBR322质粒的特定位点(3 195,3 222或3 281位点)。应用稳态和时间分辨荧光法研究十字形结构DNA的形成。结果显示:(1)稳态荧光特性表明,当PdC掺入到pBR322质粒的3 222位点,PdC-pBR322超螺旋荧光强度大约是松弛型PdC-pBR322的4倍;与此同时,其时间分辨荧光寿命大约比松弛型PdC-pBR322长约0.3 ns。当PdC掺入到3195或3281位点时,稳态荧光光谱和荧光寿命(两位点处约1.42 ns)并没有显著变化。(2)随着盐浓度的增大,荧光寿命略微有变化,但不是很明显。通过检测和分析pBR322质粒的荧光光谱和荧光寿命,表明能够形成在非配对环状部分3222位点含有dC的十字形结构。在一定的盐浓度(0-100 mmol/L)条件下,十字形结构能够保持其稳定性。

关键词: 十字形结构, pBR322质粒, PdC, 稳态和时间分辨荧光光谱法

Abstract: The specific aim of this work is to probe the formation process of DNA with cruciform extrusion in a pBR322 plasmid using spectroscopic methods while without digesting DNA into pieces. Pyrrolo deoxycitidine(PdC)was incorporated into pBR322 to substitute for dC at specific sites of 3 195,3 222 or 3 281. Steady-state and time-resolved fluorescence were employed to examine the specific properties of PdC in the cruciform region. Results were as followings:1)Steady-state fluorescent properties indicated that the fluorescence intensity of supercoiled PdC-pBR322 was about 4-fold stronger than that of relaxed PdC-pBR322,when PdC was incorporated into pBR322 at position 3 222;meanwhile,its time-resolved lifetime was about 0.3 ns longer than that of relaxed PdC-pBR3322. When PdC was incorporated at position 3 195 or 3 281,there was no significant change in either steady-state fluorescence spectra or time-resolved lifetime(about 1.42 ns at both positions). 2)Lifetimes changed a little with the increasing of salt concentration,but not significantly. In conclusion,by analyzing fluorescence spectra and lifetimes(both in relaxed state and supercoiled plasmid),a cruciform structure with dC at site 3 222 of impaired loop is formed,and the cruciform is stable within this investigated salt concentration range(0-100 mmol/L).

Key words: cruciform extrusion, pBR322 plasmid, PdC, steady-state and time-resolved fluorescence spectroscopy