I'm trying to add Trifluoroethanol (TFE) to a DNA oligo to induce the A-form. Apparently, the reaction is supposed to saturate at 80% TFE, however my oligo appears to precipitate with the addition of >50% TFE. The addition of 50% TFE increases the ellipticity at 260 nm so I suppose this means that the TFE is working. I prepare samples by adding pure TFE to my DNA in buffer, to the final desired concentration. Can anyone tell me why the DNA is precipitating, or if I'm doing something wrong in my preps?
To induce the A form and avoid precipitation needs to use very low ionic strength. We usualy start with DNA dissolved in 1 mM phosphate buffer + 0,3 mM EDTA (the presence of EDTA is also important) and then add ethanol or TFE containing also 1 mM phosphate. The increase of ellipticity at 260 nm at 50 % TFE rather indicates psi aggregatin. The other verification of A form is a negative CD band at 210 nm }see our paper in NARes 27 (1999) 3466-3473. Good luck, Michaela
Michaela, I have indeed read a couple of your published works. I think my DNA precipitates because the TFE did not have any buffer salts in it. Thanks. :)
Also, my buffer is composed of 150mM NaCl, and 10mM HEPES, pH 7.5, perhaps the salt levels are too high? at exactly 50% TFE, the DNA does not precipitate, and the 260nm peak increases.
The low salt conditions are crucial to omit aggregation.TFE does not need to contain buffer. I have added it only to preserve the ionic strength constant during alcohol addition. M.
Michaela, I added 0.3mM EDTA and made up a mix with 1mM HEPES and 15mM NaCl (diluted the stocks we had) and observed no precipitate, with a beautiful A-DNA profile at up to 84% TFE! Thanks so much for your advise!
Benedict
Hi Benedict, I am very glad that you have succeeded. My congratulations! Michaela
Michaela, I was wondering if it would be correct to assume that since my sequence has poly-dG tracts that the dominant positive peak at 263 nm can be attributed to my oligo having an A/B form?
Hi Benedict, is the complementary strand present in your solution? If not, then the spectrum is surprising as it perfectly corresponds to the A form or to an RNA molecule. Michaela
The black trace in the figure above is indeed a duplex of 5'-CGGACAAATACCCCTGGTGGGTATATATGG-3', I'm inclined to believe that the G-tracts/C-tracts as well as the poly-d(TA) repeats are the ones contributing to the signal at 263.5, where the peak minimum at 245 is from the heterogeneous nucleotides as well as B-DNA characteristics of the poly-d(G) and poly-d(TA) duplexes.
That being said, would this alone be convincing to say that such elements certainly contribute to the peak at 260 nm or perhaps I must use a control wherein the G-tracts are scrambled, or mutated into A-tracts?
Eventually though, the goal would be to be able to discern whether the addition of protein can cause an increase at 260 nm, and if that could be attributed to the manipulation of possibly a B/A-intermediate DNA into more A-like characteristics
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