High fidelity thermostable DNA polymerase is a key requirement for all kinds of PCR applications. In our lab, Finnzyme's Phusion DNA polymerase was bought for cloning lengthy and difficult DNA strands. We have found the enzyme to be highly robust and very accurate in our experiments.
The enzyme offers a very high processivity. We have successfully amplified a 2 kb gene in 45 seconds. We have mainly used this enzyme for site directed mutagenesis and have amplified plasmids of around 7.8 kb with mutant primers. The entire PCR reaction with 25 cycles generally gets completed in about 2 hours as compared to about 5 hours with other enzymes. The sequences of the desired mutants obtained using Phusion DNA polymerase have been confirmed by DNA sequencing reactions.
The enzyme possesses both the 5' to 3' polymerase activity for DNA synthesis and a 3' to 5' exonuclease activity for proofreading purposes. Though the exonuclease activity can lead to the formation of degraded primers during the process of setting up of the reaction, lowering of the temperature to about 4 C significantly reduces primer degradation. The enzyme generates perfect blunt end products in a PCR reaction, which, upon purification, can be used directly in blunt end ligation reactions (with the aid of blunt end ligation kits) or through A-tailing in a T-based vector ligation.
Phusion DNA polymerase is shipped with two types of buffers. The 5x Phusion HF buffer can be used for routine applications. Although we obtained good results with HF buffer even in the case of GC rich sequences, the company also provides 5x Phusion GC buffer for cases in which the HF buffer does not work efficiently. Both buffers are optimized for almost all PCR applications, however, a stock of 50 mM magnesium chloride is also provided for Mg optimization in the reaction. A vial of DMSO is also included for certain PCR reactions. A brochure containing detailed information on the salient features of this polymerase as well as valuable suggestions for setting up PCR reactions using this product is also provided.
The main drawback of the enzyme, however, is in terms of its cost. It is quite expensive, amounting to nearly double the price of other proof-reading enzymes available in the market. Nevertheless, owing to its extreme usefulness in routine cloning as well as advanced PCR applications, and the fact that the enzyme requirement per reaction is minimal, the cost–to–benefit ratio is rather well balanced compared to other products.
It is a worthy investment for molecular biologists who desire to carry out multiple mutations with high fidelity and/or amplify long templates, especially GC rich sequences, all at short PCR times.
Molecular Biophysics Unit
Indian Institute of Science