Estimation of total DNA in crude extracts of plant leaf tissue using 4',6-diamidino-2-phenylindole (DAPI) fluorometry.
A method of estimating total double-stranded DNA in crude extracts of citrus leaf tissue by evaluating the enhancement of fluorescence intensity of 4',6-diamidino-2-phenylindole (DAPI) was assessed. For pure citrus DNA and citrus leaf tissue crude extract each in the presence of 100 ng/ml DAPI, excitation spectral response curves converged at excitation wavelength of 360 nm. At this excitation wavelength, maximum fluorescence intensity occurred across a range of emission wavelengths from 445 nm to 460 nm. The appropriate excitation and emission wavelengths were shown to be 360 nm and 450 nm, respectively. Fluorescence intensity increased linearly with DNA concentration and non-DNA components of the tissue homogenates had negligible effect on fluorescence at these wavelengths. Sodium dodecyl sulfate (SDS) in the incubation solution resulted in some suppression of DAPI-DNA fluorescence and produced a non-linear response to changing DNA concentration. The method should be applicable to DNA quantitation from crude tissue extracts of any plant species.
Lee
L S
LS
Queensland Department of Primary Industries, Bundaberg Research Station, Australia.
Garnett
H M
HM
D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D007211:Indoles; D010936:Plant Extracts / Q000737:chemistry*; D010944:Plants / Q000235:genetics
D005456:Fluorescent Dyes; D007211:Indoles; D010936:Plant Extracts; C007293:DAPI; D004247:DNA