IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

pfu and Taq PCR mutation percentage

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Jan 11 06:10:29 EST 2000


In article <85eh2u$78b$1 at ssauraab-i-1.production.compuserve.com>, Gys de
Jongh <GysdeJongh at compuserve.com> writes
>
>Dr. Duncan Clark <Duncan at nospam.demon.co.uk> wrote in message
>news:9erByuAEgxd4EAXA at genesys.demon.co.uk...
>>
>> Mutation frequency for Taq is 8 x 10E-6/bp/duplication
>> Mutation frequency for Pfu is 1.3 x 10E-6/bp/duplication
>
>Should this be the number of wrongly incorporated bases after the
>duplication of one base (?) Is the dimension than 8x10E-6base/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%.
>
>Can you explain this multiplication ?
>The outcome seems to be 0.48 ; why the 48% ?

All right the fraction of the total with an error is 0.48 therefore in
percentage terms, 48% of the total will have an error.

>If you multiply the chance for incorporating one wrong base (8x10E-6) with
>the total number of bases than this will yield the number of wrong bases.
>However this will be 8x10E-6 x 3000bp x 2^20 (why 20 ?).
                                                  ^^^
Because 20 cycles is in theory 20 duplications but why are you doing
2^20 when the error rate is given per duplication, which I assumed
covered the 2^ part?

Assuming the error rate is 8 x 10E-6/bp/duplication I read that as for
every base duplicated the error rate is 8 x 10E6. So if you do one
duplication of 3000bp then the fraction with errors will be 3000 x 8 x
10E-6 or 0.024. Therefore x 100 to get to % =2.4%. 20 duplications then
gives 48%. 

> If you want to
>express this as a fraction than you will ofcourse get the original 8x10E-6
>back (?)

This was done from memory from an article I think in Stratagene's
Strategies vol 12 no. 1 (which is buried in the lab somewhere) but I am
pretty sure I got it right from their article. I gave up maths a long
time ago so leave it those who are better qualified to comment further. 

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!

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




More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net