Overhang A of PCR

Paul N Hengen pnh at ncifcrf.gov
Thu May 1 12:49:36 EST 1997


Dr. Duncan Clark (duncan at genesys.demon.co.uk) wrote:

> I have had several emails about the list I posted and if people are
> interested I am quite happy to collate a list of all pols available. I
> can only do this if I'm emailed with omissions. If Paul Hengen is
> agreeable then maybe this can be added to the FAQ.  

Duncan:

There has been a list of polymerases and reported fidelity circulating around
from previous discussions here. Maybe you would like to update it to include
the newest enzymes and report whether they have exonuclease activity, etc.
This is something I've been meaning to do for some time and the FAQ list does
need to be updated. It's unfortunate that I might not be able to do it before
I leave my current position.

Here is the previous posting that I grabbed from
http://research.nwfsc.noaa.gov/protocols/taq-errors.html

--

U.S. Dept Commerce/NOAA/NMFS/NWFSC/Molecular Biology Protocols

Error Rates for Thermal Resistant DNA Polymerases

This list was originally compiled by Eric First (erfi at eel.sunet.se) and
later posted to the bionet.molbio.methds-reagnts newsgroup by Paul Hengen
(pnh at fcsparc6.ncifcrf.gov). Except where indicated, errors/bp indicates
total errors (e.g. base substitutions, frameshifts, etc.). Due to
differences in the methods used to determine polymerase fidelity it is best
to directly compare values for different enzymes only if they were assayed
by the same authors. Vent, Deep Vent, and Pfu all possess 3'-5' exonuclease
(proofreading) activity.
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Error Rates

1.  Taq (Thermus aquaticus)
        1.1 x 10-4 base substitutions/bp   (Tindall and Kunkel, 1988)
        2.4 x 10-5 frameshift mutations/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.  KlenTaq (Thermus aquaticus, N-terminal deletion mutant)
        5.1 x 10-5 errors/bp               (Barnes, 1992)

3.  Vent (Thermococcus litoralis)
        2.4 x 10-5 errors/bp               (Cariello et al., 1991)
        4.5 x 10-5 errors/bp               (Ling et al., 1991)
        5.7 x 10-5 errors/bp               (Matilla et al., 1991)

4.  Vent(exo-) (Thermococcus litoralis)
        1.9 x 10-4 errors/bp               (Matilla et al., 1991)

5.  Deep Vent (Pyrococcus species GB-D)
        No published literature.  New England Biolabs claims fidelity
        is equal to or greater than that of Vent.

6.  Deep Vent(exo-)
        No published literature.

7.  Pfu (Pyrococcus furiosus)
        1.6 x 10-6 errors/base             (Lundberg et al., 1991)

8.  Replinase (Thermus flavis)
        1.03 x 10-4 errors/base            (Matilla et al., 1991)

References:

1.  Barnes, W.M. (1992) Gene 112(1), p29-35.
        "The Fidelity of Taq polymerase catalyzing PCR is improved by
        an N-terminal deletion."
        Assay:  loss of LacZ function.

2.  Cariello, N.F., Swenberg, J.A., and Skopek, T.R. (1991) Nucleic Acids
        Res 19(15), p4193-4198.
        "Fidelity of Thermococcus Litoralis DNA Polymerase (Vent) in PCR
        determined by denaturing gradient gel electrophoresis."
        Assay:  denaturing gradient gel electrophoresis

3.  Eckert, K.A., and Kunkel, T.A. (1990) Nucleic Acids Res 18(13) p3739-
        3744.
        "High Fidelity DNA synthesis by the Thermus aquaticus DNA polymerase."
        Assay:  see Tindall and Kunkel (1988)

4.  Eckert, K.A., and Kunkel, T.A. (1991) PCR Methods Appl 1(1) p17-24.
        "DNA polymerase fidelity and the polymerase chain reaction."

5.  Keohavong, P., and Thilly, W.G. (1989) Proc Natl Acad Sci USA 86(23),
        p9253-9257.
        "Fidelity of DNA polymerases in DNA amplification."
        Assay:  denaturing gradient gel electrophoresis

6.  Kong, H., Kucera, R.B., and Jack, W.E. (1993) J Biol Chem 268(3),
        p1965-1975.
        "Characterization of a DNA polymerase from the hyperthermophile
        archaea Thermococcus litoralis.  Vent DNA polymerase, steady state
        kinetics, thermal stability, processivity, strand displacement,
        and exonuclease activities."

7.  Ling, L.L., Keohavong, P., Dias, C., and Thilly, W.G. (1991)
        PCR Methods Appl 1(1) p63-69.
        "Optimization of the polymerase chain reaction with regard to
        fidelity: modified T7, Taq, and Vent DNA polymerases."

8.  Lundberg, K.S., Shoemaker, D.D., Adams, M.W., Short, J.M., Sorge, J.A.,
        and Mathur, E.J. (1991) Gene 108(1), p1-6.
        "High-fidelity amplification using a thermostable DNA polymerase
        isolated from Pyrococcus furiosus."
        Assay:  loss of LacI (repressor) activity

9.  Matilla, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991)
        Nucleic Acids Res 19(18), p4967-4973.
        "Fidelity of DNA synthesis by the Thermococcus litoralis DNA
        polymerase--an extremely heat stable enzyme with proofreading
        activity."
        Assay:  reversion of opal suppressor in LacZ (base substitution)
                forward mutation assay (measures all mutations)
                The mutation frequencies quoted above were calculated from
                the reversion assay, so they only indicate base subtitution
                mutations.  No sequence analysis was done in this paper to
                determine the relative frequency of base substitutions and
                frameshift mutations (as was done in Tindall and Kunkel).

10.  Tindall, K.R., and Kunkel, T.A. (1988) Biochemistry 27, p6008-6013.
        "Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase."
        Assay:  reversion of opal suppressor in LacZ (base substitution)
                forward mutation assay (measures all mutations)
                sequence analysis of randomly selected mutants indicated
                that 32/42 mutations were base substitutions, 18 of which
                were T to C mutations.  Combined results of sequence
                analysis and forward mutation assay to calculate frequencies
                of base substitution and frameshift mutations.

More recently, there were several discussions in
bionet.molbio.methds-reagnts regarding the fidelity of polymerases for PCR
and isolation of home-grown Taq polymerase, which were then reviewed in
TIBS:

     P. N. Hengen, 1995. Methods and reagents - Fidelity of DNA polymerases
     for PCR. Trends in Biochemical Sciences 20 (8): 324-325

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