SSCP question (cold is better)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Fri Sep 30 14:22:44 EST 1994


Frederick C. Garbrecht writes:

> Is there any *predictable* relationship between the conformations 
> assumed by denatured ssDNA in SSCP and the gel running temperature?  
> In other words, is there any way to predict a correct temperature to 
> maximally separate conformational polymorphisms, or is it (as I 
> suspect) completely empirical?

Hi Frederick,

In theory it would be possible to make a prediction from
an elaborate computer analysis of the sequence, given that you
know the identity of the base change(s) you are detecting.  However,
I don't know of anyone who has tried this.  
So it's empirical.  There are some rules of thumb,
however.  You're trying to take advantage of low stability hairpins
that form all along the fragment and are sensitive to a base change
in the stem.  Low temp. stabilizes hairpins of lower intrinsic stability,
so there's a greater chance that your base change will find its way 
into such a stem at low temp.  So usually you have better luck at low
temp.   However, if your base change happens to be in a particularly
strong hairpin, you might have to go to higher temp. to destabilize
the hairpin with the extra mismatch.  Finally, if your fragment is large
or particulary G+C rich, then
you might have trouble with getting too many bands because there are
a lot of alternative possibilities for hairpinning.  In this case,
a higher temp. might help by eliminating the lower stability
hairpins and simplifying the pattern.

In practice, we've had better luck changing the primers than changing
the temp.  At least you can be sure the pattern is going to be 
different.

Hope this helps,
Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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