ds sequencing

Mark Guiltinan mark_guiltinan at AGCS.PSU.EDU
Tue Aug 11 01:32:50 EST 1992


In message from 8/11/92

>	I have recently isolated some positive clones from a directionally
>constructed cDNA lambda ZAP library.
...
>I am considering dsDNA sequencing.  
...
>... it produces too many GC compression artifacts),
>... would [anyone] be so kind
>as to post their favorite dsDNA sequencing method, including how they prepared
>their template and what kind of results they normally get.  I would really
>appreciate any advice (including 33-P vs 35-S vs 32-P experience).

Mike-     I have included a summary of procedures we used to sequence an
extreemly GC rich gene.  In the end, a combination of hot gels, Tdt chases and
deaza nucs. worked best.  In extreemly bad cases we broke down and used single
strand templates.

Sequenase Reaction Modifications

Conditions for sequencing close to the primer--
1.  Use 5 ug double-stranded DNA instead of 3 ug.
2.  Use 2 ul primer for annealing instead of 1 ul.
3.  Dilute G Label Mix 1:10 or 1:7 instead of the recommended 1:5.  Use 2 ul
for each reaction as usual.
4.  Limit reaction times (termination & stop) to 3 min. instead of 5 min..
5.  Add 1 ul Mn buffer before adding enzyme.


Using TDT to help eliminate pauses--
Use terminal deoxy transferase to help eliminate "pauses" by Sequenase.  Add 1
ul TDT solution to each tube (G, A, T, C) after termination step, (at same
point one would normally add STOP), and incubate an additional 30 min., 37 C. 
Then add Stop. 
TDT Solution Recipe (10ul):
1 ul10 mM dATP
1 ul 10 mM dGTP
1 ul 10mM dCTP
1 ul 10 mM dTTP
1 ul 20 units/ul TDT
2 ul Sequenase Reaction B. (5x)
3 ul water

Running Hot gels to help eliminate compressions--
Long gels (75 cm) can be run at 100 Watts and short gels (40 cm) at 70-80 Watts
if an aluminum plate is used.  Do not attempt to separate plates until they
have cooled.  Be aware that loading wells swell after prolonged heating;
consequently, load samples before swelling occurs.    No useful second loadings
can be done when running hot gels because of the gel swelling problem.


Resolving compressions by using dITP or 7-deaza dGTP for sequencing with
Sequenase--
The normal Sequenase protocol for dGTP reactions is followed except that the
appropriate dideoxy nucleotides and label mix is substituted for the dGTP
reagents.

Other sequencing modifications that we have tried--
1.  Extension reactions, following the protocol of Sequenase, did not work very
well.  Bands are faint throughout.
2.  Klenow reactions with dGTP and other dNTP's or 7-deaza dGTP as a substitute
did not work very well.  Faint bands, fuzzed bands, or no reaction at all.
3.  Sequenase reactions with Single Strand Binding Protein did not work or was
not helpful.
4.  We had greater success in eliminating compressions with Sequenase enzyme
when we used 7-deaza dGTP as compared to dITP.
5.  For annealing step, we found a 30 min. 37C incubation to work just as well
as the 2 min. 65C pulse followed by a cool-down to below 35C over 30 min..



Formamide Gels (40%) for Sequencing DNA
In some cases this can help resolve compressions.

Take 40 ml Formamide in a beaker with magnetic stir bar.
Add:	7.6 g Acrylamide
	0.4 g N-N' Methylene bis acrylamide
	42.0 g Urea
	10 ml 10X TBE Buffer
Cover with parafilm & warm in a 65C water bath (tray) with stirring until
dissolved (40-60 min.).  Mix should be about 50C.
When completely dissolved add water to a total volume of 100 ml.
Cover and continue stirring at r.t. to cool to 25C.
Vacuum filter with a nitrocellulose filter.
Add 1 ml 10% APS and 0.15 ml TEMED.  Pour immediately.  Mixture is very
viscous, so pour plates at vertical angle.  Allow 30 min. for polymerization.
Run formamide gel 60% higher than normal gel.  DNA migrates half as fast. Soak
gel in 5% acetic acid, 20% methanol for 15 min. before drying.


Also, a reposting of a comment I picked up off the usenet several months ago
from "Chris_Upton at darwin.biochem.ualberta.ca".

I once got over a REALLY stuborn compression (both directions) by doing the
sequenase labelling reaction as normal, then adding "a little" Taq polymerase,
incubating at 70C for a minute, followed by the normal termination reaction.
The extension went so far I nearly overshot what I wanted (everything had gone
to very long sequences). But it worked and I could read through the problem
area.


Good Luck

Mark Guiltinan
Penn State Biotechnology Institute
mjg at psupen.psu.edu



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