Q:Taq and deletions

Anderas Becker becker at ps1515.chemie.uni-marburg.de
Tue Nov 7 12:31:20 EST 1995


In article <47lo45$f5g at mserv1.dl.ac.uk>, "Piotr Kozlowski" <koziol at asp.biogeo.uw.edu.pl> says:
>
>Dear Netters,
>Does anyone have any suggestion on principles of generation of large 
>deletions (~500 bp) in amplificated DNA caused by the activity of Taq 
>polymerase during PCR?
>Thank you in advance for any suggestion.
>
>
>
>Piotr Kozlowski
>Institute of Biochemistry
>Warsaw University
>ul. Zwirki i Wigury 93
>02 089 Warsaw, POLAND
>Tel./Fax (+48 22) 23 20 46
>E-mail: koziol at asp.biogeo.uw.edu.pl

We had had the same problem.
It can occur if the primer has 2 anneling sites on the DNA. Only 9 bp 
at the 3'end of a primer can produce a primer shift. This is enough to amplify the new
piece of DNA instead of the expected one. This will appear if the expected 
fragment is much longer then the misannealing fragment.
If you want to exchange experiencee ask my friend Viola Frank; unpublished results-(). 
She wasted a lot of time with that problem.
Primer design is essential to avoid this problem. This was the reason why we wrote 
a new primer design program. This is really done to avoid this stupid situation.
We exclude all repeated sequences for primer design. Only unique sequences could be 
choosen to create primer.
Download the program from our ftp site, or if you use a mac try  the program amplify.
Amplify shows that missannealing before it appears in PCR. Our program tells you the 
'clean' sites. Or try any annealling check with your DNA and the primers.
If you get to know other reasons for this phenomenon, please tell me.

Andreas

----------------------------------------------------------------------
Andreas Becker
Arbeitskreis Prof. Kadenbach, FB Chemie/Biochemie, Hans-
Meerwein-Strasse, Philipps-Universitaet, 35043 Marburg, Germany
Phone: privat +49 6421 47304  Labor +49 6421 28 -5721 Fax -2191
eMail: BECKER at ps1515.Chemie.Uni-Marburg.De
WWW  : http://www.chemie.uni-marburg.de/~becker



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