Summary: Fidelity of Thermostable DNA Polymerases

erfi at eel.sunet.se erfi at eel.sunet.se
Mon Feb 21 14:49:13 EST 1994


Thanks to all who responded to my enquiry.  Values for the fidelity of
thermostable DNA polymerases are summarized below.  The methods used
to determine error rates are indicated in the reference section of
this posting.  Except where indicated, errors/bp indicates total errors
(e.g. base substitutions, frameshifts, etc.).  Please feel free to 
add to this list.

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)

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.  

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.

Eric First (erfi at eel.sunet.se)
(disclaimer: I am not associated with any of the companies selling the
enzymes discussed above)



More information about the Methods mailing list