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pfu and Taq PCR mutation percentage

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Sat Jan 8 05:57:08 EST 2000


In article <3876BCFF.A85F81AC at hotmail.com>, Liu Xiangyu
<liuxiangyu at hotmail.com> writes
>Hi, All, I am constructing a expression vecter and having a question
>about fidelity
>                 comparison of Taq and pfu polymerase
>for Taq: error rate is 8.0 X 10^-6   For pfu: ..... is 1.3 X 10^-6
>                 According to stratagene pfu DNA polymerase manual, the
>percentage of mutated PCR products after amplification of a 1KB target
>seq for 20 cycles are 2.6% for pfu and 16.0% for Taq.
>                 Does anybody knows a simple formula to caculate? for
>example, what happens to 3 KB target after 10 cycles by pfu or taq?
>                 If I use Taq for 10 cycles and pfu for 20 cycles( since
>pfu always give much weak  bands), which one gives better result
>considering percentage of mutation.
>                Thank you for your help.
>                 Liu
>


Mutation frequency for Taq is 8 x 10E-6/bp/duplication
Mutation frequency for Pfu is 1.3 x 10E-6/bp/duplication


So for a 20 cycle PCR of 3000bp target i.e. 1,000,000 fold
amplification, errors introduced by Taq are (8 x 10E-6) x (3000bp) x
(20) duplications or 48%. For Pfu in the PCR the errors would be 7.8%.
For a Taq/Pfu mix with a rough error rate of 3.5 x 10E-6/bp/duplication
the errors would be 21%
  
For 10 cycles Taq would give 24%, Pfu 3.9% and a mix 10.5%. 

The actual result for would be to give less errors because this
calculation is assuming 100% efficiency of amplification. For 20 cycles
you may be actually getting only 16/17 duplications etc.

So to reduce errors, use as few cycles as possible (therefore as much
template as possible) and a proof-reading mix.



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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