DNA fragmentation after long treatment with alkali

[David F. Spencer]

In article <ari9td$878$1 at sun250.unimi.it>, "Fabio" 
<fabio.colombo at unimi.it> wrote:

> Please does anyone have some references about DNA fragmentation after
> treatment with alkali foor long time (12 h-days)?
> thankyou in advance
> Fabio
> 
> 

DNA is chemically very stable in alkali. The N-glycoside linkage for A 
is the most labile, next for C. However it takes fairly severe 
conditions to cause the effects, just look at the reaction used in the 
original Maxam and Gilbert method for chemical sequencing of DNA. There, 
DNA is incubated in 1.2N NaOH at 90C for about 10 minutes and that gives 
about one (or fewer) cleavages per 400 or so nucleotides. Extrapolate 
back to room temperature assuming a Q10 of 2 (presumably a reasonable 
estimate) and you can see that (as a ball-park figure) you would predict 
about 1 lost A per 400 nukes per 21 hours in 1.2N NaOH.

Single lane DNA sequencing methods employ piperidine (say 0.5M, which 
would give a pH of about 11) and one paper I have here incubates the 
reaction for 5 hours at 90C, this to give a readable ladder at every 
position, and certainly far short of full hydrolysis.

The breakage of an N-glycoside linkage would eventually lead to a break 
in a phosphodiester bond, hence a single strand break. The DNA would 
obviously be single-stranded under the high pHs so double-strand breaks 
are irrelevant.

Dave

-- 
David F. Spencer, PhD
Dept. of Biochemistry and Molecular Biology
Dalhousie University
Halifax, Nova Scotia, Canada

DSpencer at Dal.CA