Characteristics of Different Nucleic Acid Staining Dyes for DNA Fragment Sizing by Flow Cytometry

• Published in: Analytical chemistry (Washington) Vol. 71; no. 24; pp. 5470 - 5480
• Main Authors: Yan, Xiaomei, Grace, W. Kevin, Yoshida, Thomas M, Habbersett, Robert C, Velappan, Nileena, Jett, James H, Keller, Richard A, Marrone, Babetta L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.12.1999
• Subjects: Chemistry; Coloring Agents; Deoxyribonucleic acid; DNA; DNA - chemistry; Flow Cytometry; Molecular Weight; Spectrometry, Fluorescence
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac990780y

An efficient and reliable double-stranded DNA (dsDNA) staining protocol for DNA fragment sizing by flow cytometry is presented. The protocol employs 0.8 μΜ of PicoGreen to label a wide range of DNA concentrations (0.5 ng/mL to 10 000 ng/mL) without regard to the solution dye/bp ratios and without initial quantification of the DNA analyte concentration. Using a combination of spectrofluorometry and flow cytometry experiments, we found that PicoGreen exhibited better overall performance than all the tested dsDNA binding dyes, such as TOTO-1. Fluorometric titration revealed that typical DNA staining protocols designed on the basis of the dye/bp ratio were highly dependent upon the DNA concentration for optimal results. PicoGreen was the least sensitive to the solution dye/bp ratio and was highly fluorescent in the presence of dsDNA. Using this new protocol, accurate histograms of HindIII digested λ DNA were demonstrated for DNA concentrations ranging from 5 to 2000 ng/mL, and for dye/bp ratios from 106:1 to 1:4 at 0.8 μΜ of PicoGreen. The new one-step protocol is broadly applicable to any sensitive, laser-induced fluorescence method for detection of nucleic acids.