Gradients with automated DNA sequencers?

Tony Murray tmurray at nyc.pipeline.com
Thu Aug 3 08:07:26 EST 1995


Dear Steve 
Resolution is a function of band separation and band width. For longer
fragments the disparity between free solution mobility and gel mobility
probably ensures good initial band width. Unfortunately band separation
gets worse for long fragments because those fragments align to the field
and look increasingly similar in mobility. This tendency can be reduced
somewhat by using reduced field gradients and/or lower gel concentrations.
The former increases elution time which generally increases band width
(diffusion) while the latter can reduce band separation. 
The most successful strategy for obtaining acceptable resolution for long
fragments unfortunately has been to run long gels to try to recover some of
the lost separation potential incurred by lowering gel concentration and to
run these gels at relatively low voltage gradients. The result is long
running times. 
Good read length can of course be obtained in shorter gels but the result
is less predictable because algorithms must then operate with low
resolution. Any additional complications for the algorithms such as low
signal to noise or substantial mobility corrections are likely to introduce
an unacceptable error rate.  
Cheers! 
Tony 
 
In bionet.molbio.methds-reagnts HARDIES at THORIN.UTHSCSA.EDU said: 
 
>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 
-- 
Tony Murray PhD. 
Research & Business Development 
Pharmacia Biotech Inc. 
800 Centennial Ave 
Piscataway NJ 08855-1327  
USA 
(908)-457-8392 (tel)  (908)-457-8246 (fax) 



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