Christian Rohrer, Eppendorf AG Introduction

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 human DNA

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 range PCR.

All PCR reactions were prepared in accordance with manufacturer protocols in 50 l volumes. Amplification was carried out in the Eppendorf M astercycler. 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
S^R = Expand 20 kb^Plus (Roche Diagnostics)
S^T = 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 polymerases

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 [3]) 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 manufact urer 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 templates

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. Fig. 3: TripleMaster PCR System in a test with a GC-rich template Conclusions

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 reactions.

Literature

[1] 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.

[2] Cheng, S., Kolmodin, L.A.; XL PCR amplification of long targets from genomic DNA. Methods-Mol-Biol. 1997; 67: 17-29.

[3] 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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