pfu and Taq PCR mutation percentage

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Mon Jan 17 06:04:47 EST 2000


In article <85go27$7pe$1 at ssauraab-i-1.production.compuserve.com>, Gys de
Jongh <GysdeJongh at compuserve.com> writes
>> Finally even if I'm out by a factor of 2 the original advice still
>> holds. Don't use Taq if you want a finished product with few errors!
>
>I can only agree here. More duplications or more error in 1 duplication will
>yield less completely right product molecules. Still the magnitude of the
>error is not what I expected. Why did I never notice that about 20% of a PCR
>product is wrong ? I did SSCP of pcr products (genomic DNA as template)
>after 35 cycles and found sharp discrete bands or shifts for point
>mutations. If 20% is wrong than I would expect smears ??? 


Sorry I haven't got back earlier. Too many other things needing my
attention.

OK so what we still need is a simple way to calculate errors and not in
the linear fashion that Stratagene erroneously published. I think that
many moons ago Boehringer published a table in one of their Biochemica's
(shortly after the launch of Expand) showing the percentage of products
with errors for Taq and Expand after 15, 20, 25 and 30 cycles and for
products from 100bp to over 1000bp. 

There is also another Boehringer Biochemica (April 95, pp 34-35) that
calculates error rates in a lacI based system for various polymerases in
PCR. Basically they used a lacI containing pUC19 (white) and PCR'd the
whole plasmid. Any errors introduced in lacI that inactivate lacI will
give blue or pale blue colonies upon ligation and transformation of the
amplified plasmid into an appropriate host. 

The error rate per bp was calculated with a re-arranged equation from
Keohavong and Thilly (PNAS 86, 9253).

f= -ln F / d x b bp

where F is the fraction of white colonies

d is the number of duplications

2(superscript)d = output DNA/input DNA

b is the effective target size of the (1080bp) lacI gene i.e. 349 bp 

There are 349 phenotypically identified single base substitutions
(nonsense and and mis-sense) at 179 codons (approx 50% of coding region)
within the lacI gene.

>From this one can calculate the error rate which for Taq comes out at
around 2.6 x 10E-5 and for Pwo (Pfu) 3.2 x 10E-6

So what's the point. Presumably with more rearrangement, assuming 20
duplications, a 3kb target size and the above error rates one can
calculate the % fraction with errors. I'll leave someone else to do the
maths!


>We also do PCR
>directly on colonies. The PCR products are cycle sequenced and analysed by
>an ABI-310. I never saw 20% noise under the peaks in the electropherograms
>????

As the errors are scattered randomly across the whole PCR fragment would
one see such a noise level as you are presumably assuming a 20% noise at
all bp?

Duncan

-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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