Gradients with automated DNA sequencers?

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Aug 2 13:24:01 EST 1995


In response to my inquirery about whether anyone uses gradient gels 
on an ABI sequencer, Tom Chappell writes: 
> 
> Why would you want to use a wedge gel in an ABI sequencer? The only point
> of a wedge is to slow down the lower MW fragments in the wedge, so they
> don't run off the bottom of the gel while you're resolving the higher MW
> fragments. On an ABI sequencer you want the fragments to fall off the
> bottom of the gel after they pass the sensor.

Thanks for your response.

Yes, all the applications of gradient gels to DNA sequencing that I
have seen are exactly as you say; and hence irrelevant to the ABI
sequencer. Theoretically, gradient gels can also deliver peak
sharpening and enhanced resolution, which could be real helpful on an
ABI sequencer, say out past 500 bases.  However, it's not trivial to
configure a gel to actually deliver this effect; and, as near as I can
tell, it may not even be possible with currently available gradient
methods, and within the practical limitations of the ABI sequencer. 
However, if my understanding is wrong and people know how to do this,
I want to know about it before I commit a blunder in this grant
proposal I'm writing.  The one gradient method that I know of that
would be technically doable on an ABI sequencer is this buisness of
putting 0.5 x TBE in the upper chamber to create a stacking gradient
to sharpen the initial peak width.  It's not obvious to me that this
actually has a discernable effect on autoradiographed sequencing gels. 
Maybe the bands stack so well to start with that there's nothing to be
gained.  Does anyone do that on an ABI sequencer; and if so, does it
do much?

Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu

For information on our graduate program in Biochemistry:
http://bioc02.uthscsa.edu/biochem.html




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