Purity of DNA

RSCOTT at IRRI.CGNET.COM RSCOTT at IRRI.CGNET.COM
Mon Jan 18 07:16:31 EST 1993


> Mitch [mitch at uniwa.uwa.edu.au]: 
> "How does the A260/A280 method of DNA concentration and purity 
> compare to the fluorometer method for these determinations.  I 
> have people swear to  me that the fluorometric method is better!"


The A260/A280 photometry is for quantitation and purity assay of 
nucleic acids in general (distinction between DNA and RNA, however,  
cannot be done here). The fluorometric assay on the other hand although 
not useful as a  gauge for protein contamination should give a more
accurate quantitation of nucleic acids. The method depends on the binding 
of a fluorescent substance (fluorochrome) to DNA. The fluorometer is 
basically similar to the UV-VIS spectrophotometer but the detector would 
be sensitive to a wavelength longer than the spectrum range of the light 
source. Specifically, the light source and detector should have 
adjustments corresponding to the maximum of the absorption and 
emission spectrum of the fluorochrome used. The most common 
fluorochrome employed for estimating nucleic acids conc. is 
ethidium bromide. But DNA assays done in solutions with EtBr just like 
the A260/A280 method would also suffer from RNA interference since 
EtBr also binds comparably to RNA. Alternatively, if the sample DNA 
is of high MW, one can do a minigel electrophoresis with the sample 
alongside standard DNA stocks followed by EtBr staining. This would 
permit resolution of contaminating RNA from the DNA. 

There are other fluorochromes like Hoechst 33258 (bis-benzimidazole) and
DAPI (diamidino-phenylindole). These dyes permit DNA-specific
assays. Both these dyes preferentially binds to DNA. RNA and protein
interference is negligibly low permitting the use of very crude
samples. The H33258 bound to DNA excites at 365 nm and emits a spectrum 
with peak at 458 nm. DAPI-DNA complex on the other hand absorbs 
maximally at 344 nm and emits at 466 nm. These dyes, however, since 
they characteristically bind A-T rich domains in the DNA would be 
inappropriate for assaying samples with very high G-C content (in that 
case, ethidium bromide which is not sentitive to base composition 
would be a useful alternative). But for routine use, H33258 and DAPI 
should give excellent readings with very fine sensitivity (as low
as 10 ng/ml DNA). Of course the choice of method would largely 
depend on your purpose.

================================================================
R.Scott
Genet. Lab.
Plant Pathol. Div.
Intl Rice Res Inst
P.O. Box 933, 1099 Manila, Philippines
e-mail: rscott at irri.cgnet.com
=================================================================




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