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Highly accurate
and easy to use
- 40
X more accurate than Taq DNA polymerase
- Easy
to use
- No
need to alter set-up conditions
- Reads
through difficult secondary structure
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Isis
is a new proofreading DNA polymerase with high-performance
features.
It is an excellent choice for PCR applications requiring very low
probability of base
mis-incorporation.
High accuracy
Fidelity studies using the highly reliable Flaman (1,2) method
showed that Isis is able to amplify DNA with 40 times more
accuracy than Taq DNA polymerase.
PCR performance of
the highly thermostable proof-reading B-type DNA polymerase from
Pyrococcus abyssi. Dietrich J et al. FEMS Microbiol Lett 2002 Nov 19;217(1):89-94
Easy to use
Isis DNA Polymerase™ shows very robust activity over a
range of different amplification conditions and does not require
primer 3'-end protection, unlike many other proofreading enzymes.
This means much less time is needed for reaction set-up and
optimization.
Highly
thermostable
Isis is the most thermostable proofreading polymerase available
and therefore will retain more activity during PCR, giving more
product. This also means denaturation time and temperature can
both be increased if necessary (see Fig.1), e.g., for reading
through difficult secondary structure.
Figure 1
Perfomance comparison of Pfu, Taq and Isis DNA
polymerases™ over a range of increasingly
challenging denaturation conditions. Amplification of a
420 bp fragment was performed using 10ng human -globin
gene as a template, 50picomoles of nonprotected primers,
100M of each dNTP and 1U of Isis , Pfu and Taq DNA pol
respectively. PCR was performed using different
temperatures of denaturation.
Standard PCR Program A: (5’ at 93C) x 1 –
(1’ at 91C, 1’ at 62C, 1’15 at 72C)
x 30
Program B: (5’ at 95C) x 1 – (2’ at
95C, 1’ at 62C, 1’15 at 72C) x 30
Program C: (5’ at 95C) x 1 – (1’ at
97C, 1’ at 62C, 1’15 at 72C) x 30
Program D: (5’ at 95C) x 1 – (1’ at
99C, 1’ at 62C, 1’15 at 72C) x 30
Molecular weight marker (MW) is pBR322 Hae III/Taq I. |
Achieving maximum
accuracy with Isis Proofreading DNA Polymerase
Maximum fidelity is achieved using the Isis incubation buffer
with dNTPs at a concentration of 40M each. Under these
conditions the probability of misincorporating a base is one per
1600 molecules of 1.0kb double stranded DNA per PCR cycle
(according to the method of Flaman et al 1,2). This corresponds
to an accuracy equivalent to that of Pfu DNA polymerase. Isis DNA
Polymerase™ is highly thermostable ; 90% of polymerase
activity remains after 5 hours incubation at 90C and 50% after
5 hours at 100C. Higher thermostability permits greater
flexibility in setting denaturation time and temperature, e.g.
for difficult, GC rich templates.
References:
(1) Flaman, J.M. et al. (1994). Nucleic Acids Research 22 (15),
3259-3260.
(2) Flaman, J.M. et al. (1995). Proc. Natl. Acad. Sci. USA 92,
3963-3967
(3) Dietrich J. et al. (2002). PCR performance of
the highly thermostable proof-reading B-type DNA polymerase from
Pyrococcus abyssi.
Applications
- PCR for
cloning
- 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
- Amplification
of templates that are GC rich, or contain secondary
structure
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Figure 2
Robust performance over a wide range of PCR conditions.
Amplification of human -globin DNA 400 bp (lane 2), 900
bp (lane 3) and mitochrondrial human DNA (4.0 kb lane 4)
with Isis DNA Polymerase™. 10 ng of each DNA
template, with 50 pmoles of each primer, 100 M of each
dNTP and 1.5 mM MgSO4.
0.5U of Isis DNA Polymerase™ was used for
amplifying 400 bp and 1U for 900 bp and 4.0 kb. |
Lane 1.
Leon™ molecular weight marker.
Amplifications programs:
400 bp 5’ at 93C (1’ at 91C, 1’ at
62C, 1’15 at 72C) x 30
900 bp 5’ at 93C (1’ at 91C, 1’ at
62C, 1’30 at 72C) x 30
4.0 kb 5’ at 93C (30’’ at 94C, 2’
at 62C, 5’ at 72C) x 20. |
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Figure 3
Thermal vents discovered 3 km below sea-level off the
Fiji Islands are populated by thermophilic bacteria that
possess highly thermostable DNA polymerases. In
collaboration with IFREMER, Qbiogene Molecular Biology is
developing and commercialising polymerases that offer
unique performance benefits for DNA amplification
applications. Isis DNA Polymerase™, originally
isolated from Pyrococcus abyssi, is the most recent
addition to the range. (Photograph Copyright Ifremer) |
Unit
definition
One unit is the amount of enzyme required to catalyze the
incorporation of 10 nanomoles of nucleosides into a DE81
adsorbable product within 30 mm under assay condition.
Storage
Buffer
20mM Tris-HCl pH 8.0, 100mM KCl,
0.1mM EDTA, 1mM dithiothreitol,
0.5% Tween 20, 0.5% Nonidet P40,
50% glycerol. |
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Incubation
buffer (1X)
20mM Tris-HCl pH 9.0, 25mM KCl,
10mM (NH4)2SO4, 1.5mM
MgSO4,
0.1% Tween 20, 0.1 mg/ml BSA. |
Quality
control
Performed on every lot of enzyme: specific PCR on genomic and
phage templates, activity check, absence of ribonucleases,
absence of nickases and endonucleases, absence of 5' exonucleases
and 5’ phosphatases, thermostability test by PCR on plasmid
DNA, purity on SDS-PAGE, absence of proteases.
Storage:
Store at -20C
Isis
DNA Polymerase™ is provided with 10 x C buffer and with a
separate tube of 30mM MgSO4.
Application
note:
 Experiment to check
yield, cloning efficiency and error rate of Isis DNA
Polymerase™.
| EPSIS100 |
Isis DNA
Polymerase™ |
100 U |
| EPSIS103 |
Isis DNA
Polymerase™ |
3 x 100 U |
Patent information
for PCR Process
Related products
Isis-&GO™ Mastermix
|
