Trace editors for the Mac and other stuff

David Johnstondaj daj at uk.ac.ic.nhm
Tue Nov 2 05:37:44 EST 1993


Sorry this is a bit long but here goes:

Bruce Roe's comments on the above yesterday, and especially the problems of 
getting longer sequences from the ABI sequencer got me thinking.

Much of our sequencing is "into the unknown"" rather than routine and 
so the rate limiting factor is not the speed of gel reading but the speed 
with which we can get the next oligo made. Hence it is vital that we 
maximise read length from any reaction set. We have extensively played 
with mannual sequencing to optimise this. Sequenase sequencing using both 
manganease and extension mix in the reaction labels well and evenly from 
approx 10 to 2000 plus bases from the primer and Hydrolink Long Ranger gels 
maintain good tight bands (albeit closely spaced) up to at least 750 bases 
from primer (best read to date).

When I was looking to select an automated sequencer, read length was a 
significant factor. Because I knew what was possible from manual 
sequencing, I asked the ABI guys about the flexibility of electrophoresis 
on the 373A and was basically told that, since the bands must pass the 
detection zone at a fixed rate (12+-2 laser scans per band), you did 
electrophoresis how they told you to do it or it wouldn't work and that was 
it. Ie. They were not interested in getting longer reads. Since, if current 
adverts are to be believed, both LKB and LiCor are way ahead of ABI on this 
score, this seemed an extreemly short sighted attitude. All power, 
therefore, to Bruce and his collegues who are trying to improve matters but 
I do wonder if there might be an "easier" way. 

Point 1: I know that reactions can be made good for a couple of thousand 
bases

Point 2: the standard ABI base calling algorithm works (or you wouldn't 
get any sequence at all)

Point 3: surely then, we should be looking not to make the algorithms even 
more complicated (and thus vunerable) than they are at the moment but to 
improve the electrophoresis conditions so as to be able to better space the 
bands that are far from the primer.

LKB make extensive use of Hydolink Long Ranger gels in the ALF which 
presumably must help acheive their long reads. The problem with 
these gels is that you usually run them more slowly than standard gels 
(hence the scans/band problem). Has anyone out there in 373A land tried any 
of the following to increase read length:

Long Ranger gels run slow or fast (by altering electrical supply and/or 
buffer concentration or gradient)

Composite gels with standard acrylamide in the detection zone (so bands 
pass through it at the correct rate) and Long Ranger above (to keep the 
bands tight), or gradients of the 2 forms of acrylamide.

or anything else on the electrophoresis front

My 2 cents worth, 


Cheers,
DAJ
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)




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