Xm. Yan et al., Characteristics of different nucleic acid staining dyes for DNA fragment sizing by flow cytometry, ANALYT CHEM, 71(24), 1999, pp. 5470-5480
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.