Lot-specific data
Tgo DNA Polymerase 03 186 202 001
Lot. No. 92489445 / 31 Oct. 2011
Vol. Act.:
1 U/␮l
0.100 ml
Endo 1:
⬎ 10 U/4 h
Endo 2:
Nick-Act.: ⬎ 10 U/4 h
RNasen:
100 reactions
⬎ 10 U/4 h
⬎ 10 U/4 h
For general laboratory use. Not for use in
diagnostic procedures. FOR IN VITRO USE ONLY.
Tgo DNA Polymerase
From Thermococcus gorgonarius
Deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7
Cat. No. 03 186 172 001
Cat. No. 03 186 202 001
Cat. No. 03 186 199 001
50 reactions
100 reactions
250 reactions
Version May 2006
Store at ⫺15 to ⫺25°C
Product overview
Volume activity
1 U/␮l as determined in the assay on activated DNA.
Origin
Tgo DNA polymerase is a new enzyme preparation
originally isolated from Thermococcus gorgonarius.
Storage and
dilution buffer
10 mM Tris-HCl, pH 8.5 (4°C), 50 mM KCl, 10 mM
2-Mercaptoethanol, 0.1 mM EDTA, 0.5% Nonidet
P40 (v/v), 0.5% Polydocanol (v/v), 50% Glycerol (v/v).
Characterization
The supplied enzyme is the recombinant form and
purified to be free of unspecific nucleases. The enzyme
has a molecular weight of about 90 kD. Tgo DNA polymerase exhibits increased thermal stability with a halflife of more than 2 h at 95 °C compared to Taq DNA
polymerase with a half-life of less than 5 min at this
temperature.
Supplied Tgo
Reaction Buffer,
5 x conc.
(including MgCl2)
50 mM Tris-HCl, pH 8.5 (25°C), 87.5 mM (NH4)2SO4,
6.25 mM MgCl2, 2.5% Tween20 (v/v), 7.5% DMSO (v/v).
Note: Storage of the buffer for a longer time at 2 to 8°C
or on ice may result in the precipitation of Mg++ ions.
Store the buffer at -15 to -25°C, thaw at 37°C and vortex carefully before use.
Proofreading
activity
Tgo DNA Polymerase is a highly processive 5´-3´ DNA
polymerase and possesses a 3´-5´exonuclease activity,
also known as proofreading activity. The enzyme has
no detectable 5´-3´ exonuclease activity.
The inherent 3´-5´ exonuclease/proofreading activity of
Tgo DNA polymerase results in a superior fidelity of
DNA synthesis compared to Taq DNA Polymerase and
other commercially available enzymes with proofreading activity.
Comparison of fidelity of different thermostable DNA
Polymerases (3)
Enzyme
Error Rate* Mutation
Frequency**
Enzyme storage/
stability
The undiluted enzyme solution is stable when stored at
–15 to –25°C until the expiration date printed on the
label.
Taq DNA Polymerase
1.3 x 10-5
5.1 %
Other commercially
available proofreading
polymerases
1.8 x 10-5 to
8 x 10-7
5.1 – 0.3 %
Tgo DNA Polymerase
4.9 x 10-7
0.2 %
* Error rate calculated according to Keohavong and Thilly (4)
**percentage of lacI- colonies
Application
0506.03120082001➃
Unit assay on activated DNA
Incubation buffer 50 mM Tris-HCl, pH 8.5 (25°C), 15 mM (NH4)2SO4,
7 mM MgCl2, 10 mM 2-Mercaptoethanol, 200 ␮g /ml
for assay on
activated DNA
BSA, 0.1 mM of each dATP, dCTP, dGTP, dTTP.
Incubation
procedure
12.5 ␮g activated calf thymus DNA and 0.1 mCi
[α-32P]dCTP are incubated with 0.01 – 0.1 U Tgo DNA
polymerase in 50 ␮l incubation buffer with a paraffinoil overlay at 72°C for 30 min. The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation followed by scintillation counting.
Unit definition
One unit Tgo DNA polymerase is defined as the
amount of enzyme that catalyzes the incorporation of
10 nmol total deoxynucleoside triphosphates into acid
precipitable DNA within 30 min at 72°C under the conditions described above.
The accuracy or fidelity with which a given DNA fragment is amplified during PCR is of vital importance for
certain experiments. For applications of PCR, where a
homogenous DNA population is analyzed (i.e. direct
sequencing or restriction endonuclease digestion), the
mutations induced by the polymerase are of little concern. However if only a small amount of template DNA
or RNA is used as starting material and if single DNA
molecules are analyzed after PCR, artifacts can be a
significant problem.
Fidelity of DNA polymerization is of particular importance for the
• cloning of PCR products
• study of allelic polymorphisms in individual RNA
transcripts
• characterization of the allelic stage of single cells or
single DNA molecules
• characterization of rare mutations in tissue
• characterization of a population of cells in culture
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Reaction conditions
General
The optimal reaction conditions depend on the template/primer pair and should be determined when setting up a reaction. The following information
summarizes the reaction conditions and most important criteria to be considered when developing a new
assay with Tgo DNA Polymerase.
Length of
fragment
Fragments up to 3.5 kb can be amplified with the standard protocol.
Amount of
template DNA
Between 5 and 200 ng of genomic DNA template can
be amplified with the standard protocol described in
this pack-insert.
Amount of
enzyme
It is important to titrate the amount of Tgo DNA Polymerase. The optimal concentration varies between 0.4
and 1.25 units per reaction, depending on the amount
of template (see Fig. 1).
Figure 1:
Lanes
1
2
Mg2+
concentration
The standard concentration of Mg2+ for Tgo DNA Polymerase is 1.25 mM and is provided in the Tgo Reaction
Buffer. Variations have little effect on the sensitivity and
specificity of the reaction.
dNTP
concentration
The nucleotide concentration should at least be 200
␮M for each dNTP. Lower nucleotide concentrations
can increase fidelity but can also activate the 3´-5´exonuclease activity of Tgo DNA Polymerase. This activity
can degrade primers and products.
The nucleotides should be added to the incubation
mixture directly before use. This prevents degradation
of deoxynucleoside triphosphates that occur at the
alkaline pH conditions of the amplification reaction.
Special PCR Grade nucleotides – that are better resistant to degradation at alkaline pH compared to other
nucleotides – are available from Roche Applied Science (see Ordering Information).
Reaction set up
In the absence of dNTPs, the 3´-5´exonuclease activity
(proofreading) of Tgo DNA Polymerase will begin to
degrade template and primer DNA. Therefore, it is
important to add Tgo DNA polymerase as the last component to the reaction mixture. This can be achieved by
using 2 separate master mixes (see standard protocol)
or by using AmpliWax. The use of AmpliWax is only
recommended for reaction volumes of 100 ␮l, for a
reaction volume of 50 ␮l do not use AmpliWax.
Primer design
The 3´-5´exonuclease activity of Tgo DNA polymerase
also acts on single stranded DNA (e.g. primers) in the
absence and presence of dNTP. This activity does usually not interfere with PCR performance but it can be
taken into consideration for primer design. To overcome slow degradation of primers, nuclease resistant*
dNTPs can be used for primer synthesis. Additionally
longer primers with maximized GC content and
focussed complementarity at the 5´-end can be advantageous.
Optimal amount of Tgo DNA Polymerase versus
amount of template
Amplification of a 1.1 kb collagen gene fragment from
different amounts of human genomic DNA template
with different amounts of Tgo DNA Polymerase.
3
4
5
6
7
8
9
10 11 12 13
14 15
16
* To prevent the digestion of PCR primers by the 3'-5' exonuclease
activity of Tgo, it might be necessary 'depending on the experiment'
to use modified oligonucleotides. It means you can use e.g. thioate
modified phosphoamitide during oligo synthesis.
Lanes 17
18
19 20
21 22 23
24 25
5 ng template
Lane 1 = 0.2 U
Lane 2 = 0.4 U
Lane 3 = 0.6 U
Lane 4 = 0.8 U
Lane 5 = 1 U
10 ng template
Lane 6 = 0.2 U
Lane 7 = 0.4 U
Lane 8 = 0.6 U
Lane 9 = 0.8 U
Lane 10 = 1 U
25 ng template
Lane 11 = 0.2 U
Lane 12 = 0.4 U
Lane 13 = 0.6 U
Lane 14 = 0.8 U
Lane 15 = 1 U
26
27
Incorporation of
modified nucleotides
Tgo DNA polymerase accepts modified nucleotides like
DIG-dUTP, Biotin-dUTP and Fluorescein-dUTP.
Carry over
prevention with
dUTP/UNG
Tgo DNA polymerase activity is inhibited by dUTP.
Therefore, it is not recommended to use Tgo DNA
polymerase in combination with dUTP/UNG carry over
prevention.
Taq DNA
polymerase
For switching from Taq DNA Polymerase to Tgo DNA
Polymerase in a given protocol: In addition to the recommendations given above, the optimal cycling conditions for Tgo DNA Polymerase – compared to Taq DNA
Polymerase – may be different. For Tgo DNA Polymerase, we recommend to lower the annealing temperature by 2-3 °C since the 3´-5´exonuclease activity
of Tgo DNA Polymerase will shorten the primer during
cycling
Lanes 16 and 27 = MWM VII
50 ng template
Lane 17 = 0.2 U
Lane 18 = 0.4 U
Lane 19 = 0.6 U
Lane 20 = 0.8 U
Lane 21 = 1 U
100 ng template
Lane 22 = 0.2 U
Lane 23 = 0.4 U
Lane 24 = 0.6 U
Lane 25 = 0.8 U
Lane 26 = 1 U
2
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Standard Protocol
Typical results
Preparation of
Master Mix 1
Figure 2:
Set up in a sterile microfuge tube on ice:
Component
Volume
Final
conc.
1 ␮l each
200
␮M
downstream primer
x ␮l
0.4
␮M
upstream primer
x ␮l
0.4
␮M
template DNA (e.g. human
genomic DNA)
x ␮l
5–
200 ng
add sterile double dist. H2O
up to 25 ␮l
dATP, dGTP, dCTP and dTTP,
10 mM each
Preparation of
Master Mix 2
Amplification of 4 fragments of increasing length from
200 ng human genomic DNA with 1 unit Tgo DNA
polymerase.
Set up in a sterile microfuge tube on ice:
Component
Volume
Final
conc.
10 ␮l
5 x Tgo Reaction buffer
Tgo DNA polymerase, (1 U/␮l)
0.4 – 1.0 ␮l
add sterile double dist. H2O
up to 25 ␮l
0.4 –
1.0 U
MWM
collagen
tPA
EPO
tPA
MWM
IV
1.1 kb
1.7 kb
1.8 kb
3.0 kb
IV
Cloning of amplified DNA fragment
Procedure
The PCR products – generated with Tgo DNA polymerase – are blunt ended and can therefore be directly
used for blunt-end ligation without any pretreatment of
the ends. Please refer to the related product section for
products - available from Roche Applied Science– that
can be used for this experiment
Please refer to the following table.
Step
Action
1
Pipet together on ice Master Mix 1 and Master Mix 2 in a PCR tube, mix and overlay with
mineral oil if required by your type of thermocycler.
2
Start cycling immediately. An example for a
cycle profile is given for the Perkin Elmer
Gene Amp 9600 Thermocycler. When using
other thermocyclers, the cycling conditions
have to be adjusted
Temp.
Time
Cycle
No.
Initial
94°C
2 min
1x
Denaturation
Denatur- 94°C
30 x
30 s
ation
Anneal- 45 - 68°C1 60 s
ing
Elonga- 72°C
45 s - 3 min2
tion
Final
72°C
7 min
1x
Elongation
Quality Control
Each lot of Tgo DNA polymerase is assayed for activity
on activated DNA. Furthermore a function test for PCR
is performed using human genomic DNA.
Each lot of Tgo DNA polymerase is assayed for contamination activities as stated below.
See data label for lot specific values.
Composition of buffer used during these experiments:
50 mM Tris-HCl, pH 8.5 (25°C), 15 mM (NH4)2SO4,
7 mM MgCl2, 10 mM ß-2-Mercaptoethanol.
Absence of nucle- 1 ␮g of λ DNA is incubated with Tgo DNA polymerase
ases
in 50 ␮l buffer at 37°C for 4 h. The amount of enzyme
units showing no degradation of lambda DNA is stated
under “Endo1“.
1 ␮g of λ DNA Eco RI/Hind-III fragments are incubated with Tgo DNA polymerase and 200 ␮M dNTPs
each in 50 ␮l buffer with paraffin-oil overlay at 72°C for
4 h. The number of enzyme units showing no alteration
of the banding pattern is stated under “Endo2“.
1 annealing temperature depends on the melting
temperature of the primers used.
2
elongation time depends on the length of the fragment to be amplified. Use 45 s for targets up to 1 kb,
use 1 min for fragments up to 1.5 kb and 2 min. for
fragments up to 3 kb.
Absence of
1 ␮g of supercoiled pBR322 DNA is incubated with Tgo
“Nicking activity” DNA polymerase with 200 ␮M dNTPs each in 50 ␮l
buffer at 37°C for 4 h. The number of enzyme units
showing no relaxation of supercoiled DNA is stated
under “Nick-Act.“.
3
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References
1 Thermostable nucleic acid polymerase from Thermococcus gorgonarius, WO 9814590
and EP 0 834 570 A1 patents
2 Hopfner et al., (1999) PNAS, 96, 3600-3605
3 Frey B. & Suppman B. (1995) BIOCHEMICA, 2, 34-35.
4 Keohavong, P. and Thilly, W.G. (1989) PNAS, 86, 9253
Ordering Information
Product
dATP, PCR-Grade
Pack Size
25 ␮mol (250 ␮l) –
100 mM
125 ␮mol(1250 ␮l) –
100 mM
dCTP, PCR-Grade
25 ␮mol (250 ␮l) –
100 mM
125 ␮mol(1250 ␮l) –
100 mM
dGTP, PCR-Grade
25 ␮mol (250 ␮l) –
100 mM
125 ␮mol(1250 ␮l) –
100 mM
dTTP, PCR-Grade
25 ␮mol (250 ␮l) –
100 mM
125 ␮mol(1250 ␮l) –
100 mM
Set of Deoxy-Nucleotide, PCR set containing 1 sepGrade
arate vial (25 ␮mol –
250 ␮l, 100 mM) of
each nucleotide
PCR Nucleotide Mix
200 ␮l – for 100
reactions
Premixed ready-to-use solution of PCR Grade deoxynucle- 10 vials of 200 ␮l –
otides at a concentation of 10
for 1000 reactions
mM
Rapid DNA Ligation Kit
1 kit for 40 reactions
Shrimp Alkaline Phosphatase
1000 U
PCR Blunt End Cloning Kit
1 kit for 40 reactions
Cat. No.
11 934 511 001
11 969 013 001
11 934 520 001
11 969 021 001
11 934 538 001
11 969 030 001
11 934 546 001
11 969 048 001
11 969 064 001
11 581 295 001
11 814 362 001
11 635 379 001
11 758 250 001
11 939 645 001
Roche Applied Science offers a large selection of enzymes, reagents, and
systems for PCR and RT-PCR assays. For a complete overview, please visit
and bookmark our Amplification Special Interest Site at http://rocheapplied-science.com/PCR
Trademarks
AMPLIWAX is a trademark of Roche.
Licensed by New England Biolabs (NEB) under U.S: Patents Nos. 5.322.785,
5.352.778, 5.500.363 and foreign counterparts.
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