103
Name of enzyme and description
Special characteristics
Source
Applications
References
Sequenase: Modified T7 DNA polymerase with
low 3′
→ 5′ exonuclease activity created by an
iron-catalyzed oxidation reaction. A mutant with
nil exonuclease activity but enhanced polymerase
activity has led to version 2 of Sequenase.
Exonuclease activity is
greatly reduced.
Bacteriophage T7
Sanger DNA sequencing.
Tabor and
Richardson
(1989)
TaqDNA Polymerase: Most well known among
DNA polymerases and is responsible for
transforming PCR into an indispensable tool for
molecular biology. This 94-kDa polypeptide has a
5′
→ 3′ structure-dependent nuclease activity in
addition to its 5′
→ 3′ polymerase activity.
Nontemplate-dependent
terminal transferase
activity Lacks 3′
→ 5′
exonuclease activity
Thermus aquaticus
PCR, TA cloning which uses the
complementarity between a single
3′-T overhanging of a linearized
‘T-vector’ with PCR products
having 3′-A overhangs
Brock and
Freeze (1969),
Zhou and
GomezSanchez (2000)
PfuDNA Polymerase: A 90 kDa thermostable
DNA polymerase isolated from a
hyperthermophilic archaebacteria. It exhibits a
long half-life and higher stability than Taq
polymerase.
3′
→ 5′ exonuclease
activity and a high
proofreading efficiency
Pyrococcus furiosus DNA polymerization reactions
requiring high- fidelity synthesis
such as gene cloning, gene
expression, or mutation analysis
Angers et al.
(2001)
Vent DNA Polymerase: Also known as Tli
DNA
polymerase, it is a 93 kDa high-fidelity
thermostable DNA polymerase from a
hyperthermophilic archaebacteria.
3′
→ 5′ proofreading
exonuclease activity
Thermococcus
litoralis
PCR
Belkin and
Jannasch
(1985)
High thermostability with
a half-life of 8 h at 95 °C
and about 2 h at 100 °C
Primer extension
Deep Vent DNA Polymerase: High-fidelity
thermophilic DNA polymerase from a Pyrococcus
sp. isolated from a submarine thermal vent at
2010 m with temperatures as high as 104 °C.
3′
→ 5′ proofreading
exonuclease activity
Pyrococcus
sp.
GB-D
PCR
Jannasch et al.
(1992)
High stability with half-life
of about 24 h at 95 °C.
Primer extension
(continued)
4 Enzymes as Molecular Tools
Name of enzyme and description
Special characteristics
Source
Applications
References
Sequenase: Modified T7 DNA polymerase with
low 3′
→ 5′ exonuclease activity created by an
iron-catalyzed oxidation reaction. A mutant with
nil exonuclease activity but enhanced polymerase
activity has led to version 2 of Sequenase.
Exonuclease activity is
greatly reduced.
Bacteriophage T7
Sanger DNA sequencing.
Tabor and
Richardson
(1989)
TaqDNA Polymerase: Most well known among
DNA polymerases and is responsible for
transforming PCR into an indispensable tool for
molecular biology. This 94-kDa polypeptide has a
5′
→ 3′ structure-dependent nuclease activity in
addition to its 5′
→ 3′ polymerase activity.
Nontemplate-dependent
terminal transferase
activity Lacks 3′
→ 5′
exonuclease activity
Thermus aquaticus
PCR, TA cloning which uses the
complementarity between a single
3′-T overhanging of a linearized
‘T-vector’ with PCR products
having 3′-A overhangs
Brock and
Freeze (1969),
Zhou and
GomezSanchez (2000)
PfuDNA Polymerase: A 90 kDa thermostable
DNA polymerase isolated from a
hyperthermophilic archaebacteria. It exhibits a
long half-life and higher stability than Taq
polymerase.
3′
→ 5′ exonuclease
activity and a high
proofreading efficiency
Pyrococcus furiosus DNA polymerization reactions
requiring high- fidelity synthesis
such as gene cloning, gene
expression, or mutation analysis
Angers et al.
(2001)
Vent DNA Polymerase: Also known as Tli
DNA
polymerase, it is a 93 kDa high-fidelity
thermostable DNA polymerase from a
hyperthermophilic archaebacteria.
3′
→ 5′ proofreading
exonuclease activity
Thermococcus
litoralis
PCR
Belkin and
Jannasch
(1985)
High thermostability with
a half-life of 8 h at 95 °C
and about 2 h at 100 °C
Primer extension
Deep Vent DNA Polymerase: High-fidelity
thermophilic DNA polymerase from a Pyrococcus
sp. isolated from a submarine thermal vent at
2010 m with temperatures as high as 104 °C.
3′
→ 5′ proofreading
exonuclease activity
Pyrococcus
sp.
GB-D
PCR
Jannasch et al.
(1992)
High stability with half-life
of about 24 h at 95 °C.
Primer extension
(continued)
4 Enzymes as Molecular Tools