ABI sequencing and Stratagene Taq Extender

Duncan Clark Duncan at genesys.demon.co.uk
Mon Oct 3 03:51:11 EST 1994


In article: <anderson-290994104324 at 128.252.181.14>  anderson at pharmdec.wustl.edu (Eric C. Anderson) writes:
> 
> okay, here's an interesting question for those using the ABI Taq Terminator
> sequencing kits.  
> 
> as most of you know, ABI claims that the Taq Terminator chemistry is good
> for about 400bp of sequence.  i've been able to bump it up to 500bp with
> good template and primer combinations (and a little voodoo chant i learned
> down in New Orleans, but never mind that).  what i'm wondering is if anyone
> has used the Taq Extender from Stratagene to try to increase the read
> distance.  from what i understand of both products, there isn't anything
> obvious that would make one or the other of them screw things up, and it
> seem that if it worked (i.e. gave another 100bp or so of good sequence) it
> would be a cheap and easy way to stretch the sequence, as opposed to
> switching to the Dye Primer or Sequenase chemistry kits.

There was a thread similar to this a few weeks back but results never 
appeared. Basically taq extender is dilute Pfu approx 1/16. Pfu has a 
3-5' exonuclease and as such will remove mis-incorportaed bases 
including ddNTP's. This used to be a standard test for whether your 
unknown pol had proof-reading exo or not. Given that the Pfu is going 
to be used in such a diluted amount the original thread pondered 
whether it would remove the ddNP's or not. I reckon that in order not 
to remove the ddNTP's one would have to use so little Pfu that it's 
polymerase effect would be negligible. It works for long PCR because 
you want to remove mis-incorporated bases or depurinated bases. 
Cetus reckon the best comnbination for long PCR is TthHB8 plus Vent 
'cos Vent has high affinity for DNA and low processivity ie 
Vent hops on, repairs the mistake and hops off again allowing the 
TthHB8 to continue extending.

Anyway try and please report back. Just rember to be prepared to 
use v.low amounts of Taq extender or Pfu.

Duncan    
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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
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