稳态和时间分辨荧光光谱法检测pBR322质粒中十字形结构

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¹，²,张旭²，³

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 Lan¹，² ,ZHANG Xu²，³

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/Abstract

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

夏岚,张旭，. 稳态和时间分辨荧光光谱法检测pBR322质粒中十字形结构[J]. 生物技术通报, 2016, 32(4): 210-216.

XIA Lan， ,ZHANG Xu，. Steady-state and Time-resolved Fluorescence Spectroscopy to Detect the Cruciform Structure in pBR322[J]. Biotechnology Bulletin, 2016, 32(4): 210-216.

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