Error rate in Taq pol. 1:270?
Dr. Duncan Clark
duncan at genesys.demon.co.uk
Wed Feb 12 04:38:19 EST 1997
In article <33015E83.1737 at bcc.orst.edu>, "Bryan L. Ford"
<fordb at bcc.orst.edu> writes
>> 30 cycles of exponential amplification will give 2 to the power of 30
>> fold synthesis ie 1,074,000,000 x 810bp.
>
>And unfortunately there is a considerably weakness in the above
>statement. It is safe to say that one *never* sees anything near the
>theoretical "2 to the nth" number of products. There may be several
>reasons for this, but one reason is that not all templates productively
>participate in each round of PCR. In analyses of this, one often sees
>terminology such as "effective cycle number", that is "2 to the 30th"
>might be adjusted to "2 to the 18th" based on empirical determination of
>the number of product strands.
Hi Bryan,
I agree there is a weakness in the 2 to the nth because that is the
theoretical maximum and we all know that this is never reached. I've
seen the odd mention of say an efficiency factor (for lack of an
alternative name) of 0.6. For some work we have been doing on fidelity,
18 cycles only gives an effective amplification equivalent to roughly 8
duplications. However we are amplifying approx. 4.5kb. For a 500bp
target amplified for 20 cycles we see around 17 duplications.
Whatever the actual no. of duplications is, the base substitution error
rate per cycle for Taq is well documented as around 1 in 9000. This has
been published by Wayne Barnes, Eckert and Kunkel and Tindall and
Kunkel. The one-base frameshift error rate is of the order of 1 in
41000.
At the end of the day you are going to get errors using Taq. The only
real solution is a proof-reading pol but you will have to trade off
yield. If you use Taq for cloning then use the least no. of cycles
possible and start with as much template as possible.
Duncan
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Duncan Clark
DNAmp Ltd.
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