error rate of polymeases
Dr. Duncan Clark
news at hgmp.mrc.ac.uk
Tue Apr 10 05:07:43 EST 2001
In article <200104100326.f3A3Q3814223 at ibms.sinica.edu.tw>, the eminent
Wolfgang Schechinger at Academia Sinica wrote
>To my knowledge, the error rate of Taq is said to be 1/1000 ,
>that of proofreading Pols (Pwo, Pfu) ist said to be 1/5000.
Depends on who you quote :-(
Tindall and Kunkel in Biochemistry Vol 27, pp6008-6013 (1988) reported
that Taq produces single base substitution errors at a rate of o for
each 9000 nucleotides polymerised. Frameshift errors were 1 in 41000.
>Are these figures close to reality and what do they actually
Very dependnat upon reaction conditions in the PCR. Eckert and Kunkel in
NAR, Vol 18, pp3739-3744 (1990) reported up to ten fold lower error
rates if one uses equimolar concentrations of dNTPs and MgCl2.
>When I copy a gene with Taq,
>there will be (statistically) 1 error in every 1000 bases?
Every one thousand bases polymerised - yes. i.e. potentially two errors
in PCRing 1000bp with one cycle, one per strand.
>Meaning that after a 30 cycle (theoretical) PCR starting from a
>single copy, there should be 30 errors within 1000 bp (after
Sounds reasonable but should it be 60? Whatever, theoretically, it is a
lot of errors :-( However we have never seen anything like that many
errors but we tend to use nor more than 15-20 cycles if cloning the
product for expression. Plus we use proof-reading either by a mix or
with say just Pfu.
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR:
Optimisation, Probe Technology & Future Systems
4-5 September 2001
King Alfred's College, Winchester, UK
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