PCR of GC-rich genes

John W. Longshore Gene009 at uabdpo.dpo.uab.edu
Wed Oct 26 10:22:12 EST 1994


In article <1994Oct26.125323.1 at molbiol.ox.ac.uk>, sbaker at molbiol.ox.ac.uk wrote:

> 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.  E-mail me if you need more
info or references.

Hope this helps....

            John

-- 
John W. Longshore
Laboratory of Medical Genetics
University of Alabama at Birmingham
gene009 at uabdpo.dpo.uab.edu



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