How proofreading work Re: How to avoid PCR errors?

suter at VAX.MPIZ-KOELN.MPG.d400.de suter at VAX.MPIZ-KOELN.MPG.d400.de
Wed Feb 23 04:31:25 EST 1994


bob delisio wrote (some time ago):

+Vent polymerase chews back the primers to 15 bases under normal PCR conditions.
+Thus, if your primers are 30mers with the mutation(s) somewhere in the middle
+chances are very good that you won't get the mutant you're looking for. As a
+matter of fact, all polymerases with 3'-> 5' exonuclease activity can and will
+potentially degrade primers used in PCR mutagenesis. The question becomes one
+of severity.
+
+For mutagenesis, I prefer to use good ol' Taq and adjust the conditions to
+favor a fidelitious synthesis. In general, you should:

just a side remark: i notice that you work for Hoffmann-LaRoche. are you 
allowed to use vent in a pcr reaction ?

+I should be noted that Taq tends to add non-template directed bases to the
+synthesizing strand and that the addition is preferential to a single A. Thus,
+when synthesizing a large fragment from several shorter ones, I always take
+this into account by engineering the overlap as if the A is placed there
+automatically because an A:N mismatch will almost always fail to extend in the
+next round. Since the final base in the synthesizing strand would be a A,
+the template therefore must have a T compliment:


+3' -------------------------T---------------------------------------------- 5'
+         -------------------A
+                         A------------------
+5' ----------------------T------------------------------------------------- 3'

sorry, i am a bit confused. therefore, some questions.
1. the A residue is added to the 3 prime of the DNA,is that correct ?
2. is this acivity only found for Taq, or also for all other thermo-
	resistant polymerases?
3. is it only one A ? i heard that it may be up to 3 residues, in which case
	your strategy won't be of much use.
4. if this is a terminal transferase-like activity, why doesn't taq make
	stretches of 100s of As at the end ?
5. i think the idea that you present is pretty neat, do you notice a
	difference in PCR efficiency with oligos that do not end 
	before a T in the template ? i can imagine that a large
	quantity of the primers will get an extra A after several rounds 
	of amplification, does hampering the reaction ! this could be
	a major problem.
  
cheers,
clemens
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Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10,        D-50829 Koeln, Germany
Tel.xx49.221.5062-221    Fax.-213      e-mail: suter at vax.mpiz-koeln.mpg.d400.de
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