生物技术通报2016，Vol.32Issue(4)：210-216,7.DOI:10.13560/j.cnki.biotech.bull.1985.2016.04.028
稳态和时间分辨荧光光谱法检测pBR322质粒中十字形结构
Steady-state and Time-resolved Fluorescence Spectroscopy to Detect the Cruciform Structure in pBR322
摘要
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).关键词
十字形结构/pBR322质粒/PdC/稳态和时间分辨荧光光谱法Key words
cruciform extrusion/pBR322 plasmid/PdC/steady-state and time-resolved fluorescence spectroscopy引用本文复制引用
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):210-216,7.基金项目
江苏省六大人才高峰资助项目(swyy-030) （swyy-030）
