Alkami Quick Guide™ for PCR
Taq (Thermus aquaticus)
Taq has a relatively high error rate since it does not have the 3' -> 5' exonuclease proofreading function. However, the error rate of Taq polymerase has been reduced by a factor of 2.8 (from 2 x 10-4 to 7.2 x 10-5 by modifying reaction conditions (Ling et. al. 1991).
The most common mutations observed with the use of Taq are AT to GC transitions (Keohavong and Thilly 1989). It is also highly likely to generate deletion mutations if the template DNA has the potential to form secondary structures (Cariello et al. 1991).
Notes on Taq
♦Don't store Taq diluted
♦Keep it stored in the buffer it's in
♦Pure Taq in its tube is active at room temperature.
♦Taq is shipped at room temperature, but people are told that if they receive it and forget to put it in the freezer, it's still ok to use it.
♦The dNTP's are probably more fragile than the Taq enzyme.
♦Keep Taq on ice whenever possible, especially when combined with other components.
♦Taq will extend the primer at room temperature.
Contributed by Denise Rubens, National Cancer Institute, Frederick, MD.
Suggested PCR Coreagents and Known Characteristics
dNTPs: |
40 µM - 200 µM |
Mg++: |
1 mM - 10 mM |
Salt: |
50 mM KCl |
pH: |
8.0 - 8.8 |
Buffer: |
10 mM Tris-HCl, pH 8.3 |
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Additives: BSA, NP-40, Tween 20
Error Rates:
2.4 x 10-5 frameshift mutations/bp (Tindall and Kunkel, 1988) 1.1 x 10-4 errors/bp (Tindall and Kunkel, 1988)
2.1 x 10-4 errors/bp (Keohavang and Thilly, 1989) 7.2 x 10-5 errors/bp (Ling et al., 1991)
8.9 x 10-5 errors/bp (Cariello et al., 1991) 2.0 x 10-5 errors/bp (Lundberg et al., 1991) 1.1 x 10-4 errors/bp (Barnes, 1992)
2.0 x 10-4 errors/bp
Efficiency/Cycle: 36% - 88%
Sources:
Perkin Elmer PCR Reagents catalog, "PCR Primer: A Laboratory Manual", C.W. Dieffenbach & G.S. Dveksler 1995, error rates compiled by Eric First (erfi@eel.sunset.se), Dunning et al., 1988, Keohavong & Thilly 1989
KlenTaq (Thermus aquaticus, N-terminal deletion mutant)
Klentaq is a 5'-exo-minus, N-terminal deletion of Taq DNA polymerase. It's optimal range of Mg++ concentration is broader than most enzymes so that is easier to optimize other reaction conditions.
Suggested PCR Coreagents and Known Characteristics Error Rates:
5.1 x 10-5 errors/bp (Barnes, 1992)
Sources:
Clonetech PCR Enzyme Systems brochure, error rates compiled by Eric First (erfi@eel.sunset.se)
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