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 microM 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 lambda 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 microM of PicoGreen. The new one-step protocol is broadly applicable to any sensitive, laser-induced fluorescence method for detection of nucleic acids.
Yan
X
X
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Grace
W K
WK
Yoshida
T M
TM
Habbersett
R C
RC
Velappan
N
N
Jett
J H
JH
Keller
R A
RA
Marrone
B L
BL
D004396:Coloring Agents; D004247:DNA / Q000737:chemistry*; D005434:Flow Cytometry; D008970:Molecular Weight; D013050:Spectrometry, Fluorescence
D004396:Coloring Agents; D004247:DNA