Enzyme fidelity (Re: PCR mistakes)

Tim Fitzmaurice tjf11 at cus.cam.ac.uk
Wed Jun 3 07:02:48 EST 1998


On Wed, 3 Jun 1998, Richard P. Grant wrote:

> It's a bit worrying that you're getting errors using a proof-reading
> enzyme.  I would suggest trying different enzymes.  

> Silly question - how do you know you have the mistakes?  Are you comparing
> with a published sequence?
> 

Hmm, all this discussion of numbers of mistakes makes me worry slightly. I
was trying to clone something few months ago and was having trouble, so
decided to investigate PCR as a method of generating my insert DNA. 

I went to look at Pfu, which everyone said round here was the best. The
company in question said that on 32 cycles and with a 2.5 kb fragment I
could expect on average up to 100 errors.

For this reason I discarded the idea of this approach...seeing the quoted
number of errors over the last few posts being in single figures it
appears that the figure was wildly wrong, and made the technique appear
less attractive than it really is...have I missed out here?

Tim




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