Base denaturation

David Johnstondaj daj at uk.ac.ic.nhm
Tue Oct 26 04:29:10 EST 1993


On 25 Oct 93 22:25:12 GMT,
  Dave Knecht writes:

>Hi Netters-  I have read recently several times about denaturation of dsDNA wit
>h NaOH and reference is made to the fact that the hybrids do not reform.  Does
>anyone know the basis of this statement.  I am particularly confused because we
> have always base denatured DNA before transfer to Nylon for Southerns and then
> base denatured the probe for the hybridization.  According to this dogma, the
>blot should not work, but of course it does!  Dave

Maybe there is a confusion here as to the modes of action:

NaOH is used:

(1) to denature plasmids for sequencing. I always understood that this was 
iriversible since by pulling apart the strands somewhere in a supercoil 
you get even more extensive coiling elsewhere and the plasmid basically 
tied itself into an untangleable knot with some accessible single 
stranded bits and other inaccesible "hyperknotted" bits. This would affect 
different regions on different plasmid molecules, hence there would 
always be some molecules where your primer binding site was on the 
exposed bit.

(2) In alkaline transfer to charged nylon membranes. Here you are usually 
dealing with restriction fragments, ie linear DS molecules where the 
strands separate completely on exposure to alkali and are deposited on, 
and bound to, the nylon membrane in random orientation. Once bound, there 
is no way that they are able to move around and reanneal but they are 
accesible to probes in solution. However, if the kinetics are favourable, 
a portion of an originally double stranded probe will reanneal in the 
hybridisation solution and if you don't do alkaline transfer, but 
alkali denature in the gel and then transfer with SSC, presumably a 
proportion of your target will reaneal during transfer.

Hope this is correct. Someone please shoot me down if not.

Cheers
DAJ
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)



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