Summary: PCR of GC-rich DNA

sbaker at molbiol.ox.ac.uk sbaker at molbiol.ox.ac.uk
Tue Nov 1 04:41:14 EST 1994


Here is a summary of the ideas sent to us about using high GC DNA as a 
template for PCR. We didn't say, but the product was from a defined region, ie 
to subclone an intergenic region, so the "pick a low gc area to prime from" 
posts missed the mark a little. We have no problem doing PCR up to 5kb 
normally on the same organism, using 50% GC primers and Taq Extender.


>In our lab we have a gene that is 73% GC, more or less across the whole of the
>gene. This means that primers have a melting temperature of around 70-75
>degrees C, even for 17 to 18 mers. We have had no success from amplification.
>
>Does anyone have any suggestions for PCR conditions/protocols for
>amplification of a 900-1300bp fragment?
>
>Simon Baker
>Dudley Page
>Oxford, UK


   Try using deaza dGTP in your nucleotide mix.  Start with a 75% dGTP/25%
deaza dGTP mixture with normal dATP, dCTP, and dTTP concentrations.  The
only disadvantage to this procedure is that the product will not stain
well with ethidium bromide.  Therefore, you must visualize the produce
using radioactivity that you incorporate or using labeled primers.  The
deaza dGTP method has been used successfully to amplify across high GC
regions for several years in our laboratory.

John W. Longshore
University of Alabama at Birmingham


You might try including 10% DMSO in the PCR.  We found this was required
to amplify a GC rich alternative exon in Drosophila Troponin I.

Cliff Beall
Ohio State University


          You might wish to consider the use of 7deaza2deoxyguanosine
          in your PCR. The reference for this is NAR 16:9869 (1988).

Doug McKenzie
Stratgene


Try adding 10% glycerol (the Herpes lab next store has to do this because the
Herpes genome is 70% GC!) to the rxn

Margret Shirra
Harvard


I've had good luck amplifying GC rich sequences from M.paratb. (see Vary et
al, J.Clin.Micro 28:933-937, 1990) but the shorter the better. Besides
mycobacteriologists, other people with related problems include those trying
to amplify long cgg repeats in affecteds with fragile-X syndrome (watch that
literature. You may want to try 200bp sequences for diagnostics but if you want
to isolate a big piece you may try picking primers that are as c-poor as
possible (ie whose binding sites are as g-poor as possible) and loading up on
deaza-dGTP. 

Cal Vary


Just a few suggestions:
1] Try 5-10% DMSO [fresh]
2] Try Stratagene's Pfu DNA polymerase- although you'll probably have to play
around with the buffer a bit. Pfu can withstand the higher annealing
temperatures and DMSO which tend to decrease Taq's efficiency.  We've had
excellent results with amplification of GC-rich targets by incorporating
these ideas.

M.C. Murphy


You need hot start(addition Tag before heat to 100 C after down
to primer Tm after addition Tag after run next cycle)
second suggestion is two step PCR use 94 C-72 C cycle

UNIT450 at TWNMOE10.edu.TW


I  was working on Dopamine D4 which
has a similarly (ridiculously!) high GC content. We found Stoffel fragment
of Taq was the only thing that could get PCR's to work. It's from Perkin
Elmer (no, I don't work for them!).  Hope this helps

Stuart Ball
Amersham Intermational



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