>High GC PCR

Mr. T. Weissensteiner tweissen at hgmp.mrc.ac.uk
Tue Jun 23 05:34:25 EST 1998


Dear Paul

You wrote in response to my posting on the "methods" newsgroup:

> I'm pretty sure the "GC Melt" is nothing more than TMAC. You
> should definitely look at the references cited in the article
> mentioned above, especially the one in NAR with Ken Fong as
> author...note that K. Fong is the founding father of CLONTECH ;-)

No! In their 1998 catalogue on pg. 15 they show an example of a 
PCR performed with 1.0M "GC melt". Nobody will make me believe that 
Taq polymerase is tolerating such concentrations of TMAC :-) !
The only other substance decribed to date which may (!) have a net
"GC-destabilizing" effect under PCR conditions is betaine. The
the concentration range shown to benefit  most PCRs is 0.6 - 2.0 M.

> Hung, T., Mak, K. and Fong, K. (1990) Nucleic Acids Res. 18,4953

As far as I remember they used 100 uM TMAC in a 50 mM KCl buffer.
I and others have shown that 50 mM TMACl can be employed *if* 
KCl or NH4Cl are omitted (I can dig out the references, if you are 
interested). 

> If you're using their standard buffer "X", it could cause problems
> if it contains K+. If so, you should change buffers to exclude K+.
> The following article describing this is also at my archive site:

This is exactly what I did in my buffer system (T. Weissensteiner and 
J.S. Lanchbury, 1996, Biotechniques 21, pp 1102-8). I believe the 
"problem" lies in the stabilization of template secondary structure
by salts in general (including TMACl!!) and of DNA tetraplexes by KCl 
in particular- see the papers by Woodford et al..
Some salt is certainly necessary to stabilize primer-template and 
polymerase-DNA interactions, but Mg++ or NH4+ seem much better 
alternatives.

> @article{Hengen1996Jantibs,
> author = "P. N. Hengen",
> title = "Methods and reagents - Cycle sequencing through {GC}--rich
> regions",
> journal = "Trends in Biochemical Sciences",
> volume = "21",
> number = "1",
> pages = "33-34",
> month = "January",
> year = "1996"}

Got it in my collection :-)

> The TiBS M&R article on betaine can be found on the web at:
> http://www-lecb.ncifcrf.gov/~pnh/papers/TIBS/jun97.html

This looks like a very informative site - thanks for the tip.

Regards




Thomas Weissensteiner
Autoimmunity Group
Jenner Institute for Vaccine Research
Compton / Newbury
Berkshire RG20 7NN

T     :  0044 1635 577920
FAX   :  0044 1625 577901
email :  tweissen at hgmp.mrc.ac.uk

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