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Taq errors

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Mon Sep 18 11:30:45 EST 1995

 In article <4390q4$sad at infa.central.susx.ac.uk>
 bafq5 at central.susx.ac.uk (Stephen Perry) writes:

| Does anyone have a reference or personal information about
| misincorporation rates of Taq polymerases? A comparison study would be
| most useful. Also, information about how to reduce misincorporations
| would be useful.
| Cheers
| Steve Perry.

This list was compiled by Eric First (erfi at eel.sunet.se) and posted
21 Feb 1994.  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.

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)


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-
	"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),
	"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),
	"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 
	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 here regarding the fidelity of
polymerases for PCR and isolation of home-grown Taq polymerase, which were then
reviewed in TIBS:

author = "P. N. Hengen",
title = "Methods and reagents - Fidelity of {DNA} polymerases for {PCR}",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "8",
pages = "324-325",
month = "aug",
year = "1995"}

* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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