Shearing DNA by heat - a question

David F. Spencer dspencer at is.dal.ca
Thu May 15 13:26:07 EST 1997


In article <337A1127.12F4 at unity.ncsu.edu>, Susan Hogarth
<sjhogart at unity.ncsu.edu> wrote:

> I lost the post, but someone recently described shearing DNA by
> autoclaving or boiling. How long (and at what temp) do you have to let
> it renature? Does it renature "perfectly"?

I recall that earlier thread but didn't bother responding.  Just how boiling
or autoclaving DNA will shear it is beyond me.  I assume that someone noticed
that boiled DNA runs faster on a non-denaturing gel than unboiled and somehow
made the leap to the conclusion that the DNA had gotten smaller.  Of course
denatured DNA runs faster then undenatured on non-denaturing agarose but unless 
the boiling was done at pH extremes there will be no degradation much less true 
shearing. Small, simple DNAs like plasmids reanneal fairly readily after heat 
denaturation but large complex DNAs (bacterial or eukaryotic chromosomal) don't 
reanneal unless given a long time and appropriate conditions.

Shearing DNA is a mechanical process and is only effective on ds DNA. You can
shear DNA by shooting it through syringe needles (if you like the exercise),
sonicating it (if you can stand the noise), pushing it through a French pressure
cell [this is my preference] or putting it through a nebulizer (this is a
common practice for at least some types of small genome sequencing but I've not
yet done it.)  You could approximate the result of shearing by using a 
double-stranded endonuclease, if you controlled its cutting.

You can easily confirm that heating won't shear DNA by trying it in 6X or 10X 
SSC (or 1 or 1.6M NaCl); under these conditions the Tm for 50% GC DNA should be
above 121-123 C(the temperature of standard autoclaves).

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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