染色
DNA
荧光
污渍
生物物理学
核酸
吖啶橙
化学
流式细胞术
亚历山福禄
碘化丙啶
荧光团
分析化学(期刊)
分子生物学
色谱法
生物
生物化学
光学
遗传学
物理
细胞凋亡
程序性细胞死亡
作者
Xiaomei Yan,Robert C. Habbersett,Julia M. Cordek,John P. Nolan,Thomas M. Yoshida,James H. Jett,Babetta L. Marrone
标识
DOI:10.1006/abio.2000.4789
摘要
Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.
科研通智能强力驱动
Strongly Powered by AbleSci AI