Original Repository: Alan Mason Chesney Medical Archives. Daniel Nathans Collection
Reproduced with permission of David C. Ward.
Medical Subject Headings (MeSH):
Simian virus 40
Restriction Enzymes and the "New Genetics," 1970-1980
Letter from Daniel Nathans to David C. Ward (June 24, 1971)
2nd August 1971
I'm sorry for the long delay in answering your letter of June 24th but I was out of London for the last few weeks. I would
very much like to provide you with a simple procedure to strand separate SV40 DNA. However, although we had some limited success
with the Szybalski method using poly X, we have not been able to get consistently reproducible results. The separations achieved
are extremely dependent on the poly X molecular weight (or some other unknown property). The first preparation of poly X (obtained
from A.M. Michaelson) worked nicely at the start but then deteriorated on aging. When I rechecked the S20 it had fallen from
13S to 8S. Michelson had no more poly X so I tried several commerical preps. -- none worked and none had S20s greater than
9S. I got some poly phos. from Grunberg-Manago but could not synthesize material of greater than 10S and the separations were
incomplete as judged by hybridization studies. I will include our method and early results if you should like to try the procedure
and if you can get some good poly X (which I don't have at the moment).
A number of other people have tried synthetic polymers for SV40 or Polyoma but without success (Kubinski and Rose, P.N.A.S.
57, 1720, 1967; Rust, B.B.R.C. ?, 455, 1970; Shatkin and Sambrook; Crawford and Berg (unpublished experiments). The polymers
tested include poly G, UG, IG, U, A and C. I also tried Iodo C, Bromo U, Bromo UG and diaminopurine -- all without success.
Although poly X has given some limited separation of SV40 it does not work wih polyoma DNA.
If you can find a source of very high molecular weight poly X and would like to give it a try our conditions are as follows:
1-2 [micro]grams SV40 Form II DNA in 0.1 ml of 1 x SSC + 2^-5 [micro]grams poly X. Heat at 100 degrees for 90' -- quick
cool in ice and add to CsCl gradient. A typical separation (with the original high molecular weight stuff) is shown below.
I'm sorry I can't be more helpful. Your B restriction enzyme worked fine but without the poly X step, I'm afraid
I have not been able to put it to use on the originally planned experiments.