Shearing DNA by heat - a question

David F. Spencer dspencer at is.dal.ca
Sat May 17 13:05:30 EST 1997


colossus... wrote:
> 
> David,
> 
> much as it is hard to believe, autoclaving does indeed shear the DNA.
> I prepare my sheared DNA for blocking in hybridizations this way since
> needle shearing is such a pain, and since we haven't got a sonnicator.
> How do we know it's sheared ? simple...aside from running on a gel (which
> apparently is not good enough for some), the viscosity of the DNA solution
> is decreased to such an extent that it is easy to pipette with a regular
> micropipettor. Try doing that with your non-sheared sample !!!

When DNA is denatured its viscosity drops dramatically. Double-stranded
DNA
acts (at least over short distances, say 500 bp) like a stiff rod.  
Single-stranded DNA is extremely flexible. If you wish to prove that
your 
autoclaving has merely denatured the DNA try cutting it with a four base
cutter
such as Sau3A; compare this to the unautoclaved DNA cut with the same
enzyme.
The autoclaved DNA will be virtually unaffected by a restriction enzyme
because
restriction enzymes (with a few exceptions) only degrade ds DNA and
after
denaturing, complex DNAs such as calf thymus or herring testes regain
very 
little base-pairing unless specific procedures (as used in cot curves)
are
followed.  You could also do your next autoclaving in 6X+ SSC, which
will raise
the Tm of most DNA about autoclave temperature and you will find your
"shearing"
fails.

> don't believe me ? try it yourself :) even if it were not "sheared" it
> still works beautifully in blocking blots, so I'll keep using it.
> 
> Ed
 
If you are pleased with the results of blocking membranes with denatured
DNA
that's fine; it certainly is easier to manipulate denatured DNA and it
will be
more effective than undenatured DNA.  I do take exception to the claim
that
autoclaving can shear ds DNA; it CAN'T and in fact denatured DNA is
quite
resistant (relative to undenatured) to shearing, which is a mechanical
process
requiring physical stress; the stiffness of ds DNA is what makes it so
prone to
mechanical shearing. Even before DNA gets near the 121-123 C temperature
of
autoclaves, assuming it's dissolved in water or very low salt, it will
be
completely denatured and thus essentially refractory to shearing. So
even if
you set your sonicator up in an autoclave (which might void the warranty
on the
sonicator), then went through an autoclave cycle, the DNA would be
virtually
unsheared, although denatured (and the sonicator might need some
maintenance).

Personally I don't know why anyone fools around with Denhardt's and DNA,
etc. to
block membranes during Southerns and Northerns; BLOTTO (non-fat instant
skim
milk powder with SDS) works just fine, is cheap, easy to prepare and
doesn't
need autoclaving.  For Northerns without formamide you probably should
treat the
BLOTTO with diethylpyrocarbonate to destroy RNases.  Some people use
only high
SDS concentrations as membrane blocker, a la Church and Gilbert.

Dave

===================================================================
David F. Spencer, PhD
Dept. of Biochemistry
Dalhousie University
Halifax, Nova Scotia
Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca

===================================================================



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