Vent DNA polymerase and assymmetric PCR

[Robert Horton]

molpath at chmeds.ac.nz wrote:
: In article <3ladq6$var at mule.fhcrc.org>, Tim Buss <tbuss at fred.fhcrc.org> writes:
: > Q: Will the 3' -> 5' exonuclease activity of Vent destroy the single
: > stranded product of an asymmetric PCR?
: > [deletia]
: 	Hi Tim. Could be you are right - the exo activity of Vent is probably
: 	wiping out your single-stranded product. If this is the case, what
: 	about getting some of the new 9 degrees N DNA polymerase from NEB.
: 	It is supposed to be genetically engineered to have only 1-5% of the 
: 	wild type exonuclease activity. Enough to provide proofreading, but not
: 	enough to excessivley degrade single-stranded DNA or primers.
: 	Catalog #260 (S or L).

How 'bout mixing in a smidge of proofreading enzyme with your Taq? As I
understand it, the way this is supposed to help in long-range PCR is by
having just enough exo activity to fix the errors, which increases the
efficiency of Taq's finishing long molecules. If this is so, I'd think
mixing in a bit of exo would improve the overall error rate.

Also, you want 3'-5' exo, but probably not 5'-3' exo. If mixing enzymes
works, you could pick a combination of just the right activities.


Bob Horton (Ph.D.!)   /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
St. Paul, MN 55108    ^^   horton at molbio.cbs.umn.edu/(612) 624-3790