Characteristics of different nucleic acid staining dyes for DNA fragment sizing by flow cytometry

An efficient and reliable double-stranded DNA (dsDNA) staining protocol for DNA fragment sizing by flow cytometry is presented, The protocol employs 0 .8 phl of PicoGreen to label a dde range of DNA concentrations (0.5 ng/mL t o 10000 ng/mL) without regard to the solution dye/bp ratios and without ini tial quantification of the DNA analyte concentration. Using a combination o f spectrofluorometry and flow cytometry experiments, we found that PicoGree n exhibited better overall performance than all the tested dsDNA binding dy es, such as TOTO-1. Fluorometric titration revealed that typical DNA staini ng protocols designed on the basis of the dye/bp ratio were highly dependen t upon the DNA concentration for optimal results. PicoGreen was the least s ensitive to the solution dye/bp ratio and was highly fluorescent in the pre sence of dsDNA. Using this new protocol, accurate histograms of HindIII dig ested lambda. DNA were demonstrated for DNA concentrations ranging from 5 t o 2000 ng/mL, and for dye/bp ratios from 106:1 to 1:4 at 0.8 mu M of PicoGr een. The new one-step protocol is broadly applicable to any sensitive, lase r-induced fluorescence method for detection of nucleic acids.