PCR at high altitude

Charlie Bell charlie.bell at cbr.for.csiro.au
Mon Mar 27 21:10:40 EST 1995


>Dear Netters:
>
>A quick observation (somebody correct me if this is not accurate):
>
>Taq is an enzyme, albeit one with a very high temperature optimum.
>Therefore, at a temp slightly below boiling in Quito (89 C, was it?),
>shouldn't Taq still work, even if slightly less than optimally, such that
>routine amplifications could still be accomplished? Does anyone know how
>rapidly Taq's efficiency falls off either side of 94 C? I assume the dropoff
>is steeper above 94 C?
> |===========================================|
>  David A. Watson, Ph.D. (dwatson at plains.nodak.edu)  
>  Asst. Prof. of Veterinary and Microbiological Sciences       
>  North Dakota State University 
>  ---------------------------------------------------------------------------
>                   "Some are wise, some otherwise."                           
> |===========================================|
>
>
The 94C temperature is to denature the double stranded DNA. The Taq
polymerase reaction is routinely run at about 72C. The question to be
answered is; is necessary to reach 94C to single strand the DNA? I think the
addition of salt or sugar to raise the boiling point will interfere with the
polymerase reaction. The use of DMSO sounds like the best idea to me, if
anything is necessary. Another possibility is just to increase the denature
time at a lower temperature (say 2 mins at 90C instead of 1 min at 94C). 
******************************************************
Charlie Bell              Phone 06-2818324 (International) +616-2818324
CSIRO                     Fax   06-2818312 (International) +616-2818312
Division of Forestry      E-mail charlie.bell at cbr.for.csiro.au
PO Box 4008 QVT
Canberra ACT 2600
Australia
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