Christian Rohrer, Eppendorf AG
Standard PCR components (Taq polymerase and reaction buffers) are used for
the general amplification of relatively short DNA fragments (100 bp to
roughly 2,000 bp).
However, demanding applications (such as long range PCR [1-2], high-fidelity
PCR, or the amplification of complex templates) require additional reagents
specifically designed for that application. In the past, separate kits would
have been required for different applications. The new Eppendorf TripleMaster
PCR System, however, is a universal solution for all of these applications.
Due to its unique composition of three different enzymes (Taq DNA polymerase,
polymerase enhancing factor, and proofreading enzyme) and two innovative
buffers (Tuning Buffer for long range PCR and High Fidelity Buffer
for short range PCR), the TripleMaster PCR System produces excellent results
for applications involving long range PCR, high-fidelity PCR, or amplification
of complex templates.
Methods and results
Experiment 1: Amplification of very long fragments from DNA and genomic
In order to determine the performance of the TripleMaster PCR System, a
40 kb fragment from DNA and both a 27 kb and a 24 kb fragment from humangenomic
DNA were amplified in two parallel preparations using the TripleMaster PCR
System as well as leading PCR systems that are specially designed for long
All PCR reactions were prepared in accordance with manufacturer protocols
in 50 l volumes. Amplification was carried out in the Eppendorf Mastercycler.
Result Experiment 1
Compared to all three competing products, the TripleMaster PCR System
produced markedly superior yields for all three experiments (see Fig.1).
Table 1: Final concentrations for the TripleMaster PCR System
Fig.1: The TripleMaster PCR compared to leading suppliers
in the long range PCR segment
E = Eppendorf TripleMaster PCR System
= Expand 20 kbPlus
= TaKaRa LA Taq (TaKaRa Biomedicals)
M: DNA Molecular Weight Marker XV (Roche Diagnostics)
1-3: 40 kb DNA fragment from DNA (48 kb)
4-6: 27 kb fragment of the tPA gene from human DNA
7-9: 24 kb fragment of the tPA gene from human DNA
Fragments were separated by pulsed-field electrophoresis.
Experiment 2: Comparison of the relative fidelity of thermostable DNA
To rule out the possibility of experimental conditions affecting the absolute
values of the fidelity of DNA polymerases, the accuracy of the base sequence
was determined using the IMBP (Immobilizing Mismatch Binding Protein )
method following the amplification of a 160 bp fragment.
This enables a direct comparison of polymerases under real PCR conditions
using relative data.
In three parallel preparations, a 160 bp fragment from the human gene of
the apolipoproteinB was amplified using theTripleMaster PCR System, a competitive
Taq-based product, and a Pfu-based PCR system.
All samples were prepared in accordance with manufacturer specifications.
The PCR volume was 50 l.
Result Experiment 2
The TripleMaster PCR System shows a degree of accuracy that is normally
only achieved using products that are specifically designed for high-fidelity
applications. Although the tested PfuTurbo System (Stratagene) is revealed
to have the lowest error rate, it is suitable only for the amplification
of very short sequences (< 200 bp) see Fig. 2.
Table 2: Final concentrations The new Eppendorf High Fidelity Buffer (short
range) was used in the preparation of the TripleMaster PCR System.
Fig. 2: Direct comparison of the fidelity of DNA polymerases under real
PCR conditions using relative data
Experiment 3: TripleMaster PCR System in a test with extremely GC-rich
To test the performance of TripleMaster PCR System during the amplification
of complex templates, a GC-rich (> 70 %) 490 bp fragment from the human
-actin gene was amplified.
The intron sequence between exon 1 and exon 2 of the human -actin gene
has 85 % GC content and contains stable secondary structure elements.
Table 3: Final concentrations in 50 l reaction
Result Experiment 3
Taq DNA polymerase with 2 % DMSO is unable to amplify such a complex template.
However, the TripleMaster PCR System produces very good results either in
combination with only 2 % DMSO or with only Eppendorf TaqMaster (1x) see
Fig. 3: TripleMaster PCR System in a test with a GC-rich template
The exceptionally high extension rate of the innovative enzyme mix (consisting
of high-quality Taq polymerase, polymerase enhancing factor, and proofreading
enzyme) combines with the new buffer systems (for long and short range)
to produce an impressive increase in yields for long PCR.
As the accuracy of this new system reaches a level normally reserved for
highly specific kits that can only be used for highfidelity PCR, the TripleMaster
PCR System is an all-purpose tool suitable not only for high-fidelity experiments,
but for other applications as well.
For GC-rich templates (the secondary structures which often cause complex
applicational problems) adding low concentrations of DMSO or Eppendorf TaqMaster
is sufficient to obtain very good results. Since TaqMaster, unlike DMSO,
has no negative effect on the fidelity of Taq DNA polymerase (data not shown)
it is an ideal alternative particularly for subsequent cloning and sequencing
 Barnes, W. M.; PCR amplification of up to 35 kb DNA with high fidelity
and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci.
USA. 1994 Mar 15; 91 (6): 2216-2220.
 Cheng, S., Kolmodin, L.A.; XL PCR amplification of long targets from
genomic DNA. Methods-Mol-Biol. 1997; 67: 17-29.
 Wagner, R. and Dean, A. 1998. The use of immobilizes mismatch binding
protein for optimization of PCR fidelity. In: PCR Methods Manual (Innis,
M., Gelfand, D. and Sninsky, J., eds).
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