Factors affecting quantification of total DNA by UV spectroscopy and PicoGreen fluorescence.
The total amount of DNA in a preparation extracted from tissues can be measured in several ways, each method offering advantages and disadvantages. For the sake of accuracy in quantitation, it is of interest to compare these methodologies and determine if good correlation can be achieved between them. Different answers can also be clues to the physical state of the DNA. In this study, we investigated the lack of correlation between ultraviolet (UV) absorbance and fluorescent (PicoGreen) measurements of the concentration of DNAs isolated from plant tissues. We found that quantitation based on the absorbance-based method correlated with quantitation based on phosphorus content, while the PicoGreen-based method did not. We also found evidence of the production of single-stranded DNA under conditions where the DNA was not fragmented into small pieces. The PicoGreen fluorescent signal was dependent on DNA fragment size but only if the DNA was in pure water, while DNA in buffer was much less sensitive. Finally, we document the high sensitivity of the PicoGreen assays to the detergent known as CTAB (cetyldimethylethylammonium bromide). The CTAB-based method is highly popular for low-cost DNA extraction with many published variations for plant and other tissues. The removal of residual CTAB is important for accurate quantitation of DNA using PicoGreen.
Holden
Marcia J
MJ
Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. marcia.holden@nist.gov
Haynes
Ross J
RJ
Rabb
Savelas A
SA
Satija
Neena
N
Yang
Kristina
K
Blasic
Joseph R
JR
D018744:DNA, Plant / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis*; D009930:Organic Chemicals / Q000032:analysis; D013025:Glycine max / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D003313:Zea mays / Q000737:chemistry
D018744:DNA, Plant; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen