Phusion^™ High-Fidelity DNA Polymerases:
A PCR method for amplifi cation of whole human mitochondrial DNA

In this study, the performance of fi ve different polymerases was compared when amplifying whole human mitochondrial DNA. Finnzymes’ Phusion^™ High-Fidelity Polymerases were compared to three enzyme mixes recommended for amplifi cation of long templates by the manufacturers. Phusion^™ High-Fidelity DNA Polymerases performed the amplifi cation in less time and with fewer units of enzyme than the other polymerases. Reliable amplifi cation of human mitochondrial DNA can be accomplished more quickly and cost-effectively with Phusion^™ High-Fidelity DNA Polymerases.

Introduction
Human mitochondrial DNA is a 16.6 kb circular doublestranded molecule. Mutations (deletions, duplications and point mutations) in the mitochondrial genome leading to mitochondrial dysfunction are increasingly recognized as a contributor to a wide range of human diseases. Mitochondrial dysfunction is involved in diseases such as diabetes, cancer, heart diseases and migraine. In addition, neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease are associated with mitochondrial dysfunction.

Amplifying the whole mitochondrial genome using PCR has been found to be an effi cient method for detecting mitochondrial DNA deletions involved in human diseases¹. Whereas amplifying the whole mitochondrial genome is generally useful for detecting mitochondrial deletions, that are typically relatively large, amplifying shorter target fragments is also widely used for detecting other types of mutations (e.g. point mutations) in the mitochondrial genome.

Materials and Methods
Finnzymes’ Phusion^™ High-Fidelity DNA Polymerases consist of a novel Pyrococcus-like enzyme with a double-stranded DNA-binding domain, which gives the fusion polymerase high processivity and fi delity. The performance of Phusion^™ High-Fidelity DNA Polymerases and three polymerase mixes from two other vendors were compared in amplifi cation of whole human mitochondrial DNA. Total DNA isolated from human blood was used as a template. The amplifi ed product was 16.5 kb. The primers used for the amplifi cation anneal to mtDNA at the following positions: forward 10-40; reverse 16 494-16 463¹. All reactions were conducted using conditions recommended by the manufacturers. The amounts of enzymes in units and total cycling times are shown in Figure 1. The reactions were set up on ice and run on a DNA engine Tetrad 2 thermal cycler (Bio-Rad Laboratories, Inc.).

The conditions for Phusion^™ High-Fidelity DNA Polymerase and Phusion^™ Hot Start DNA Polymerase were as follows:

The cycling conditions for Phusion^™ High-Fidelity DNA Polymerases were as follows:

Conclusions
The performance of fi ve different polymerases was compared when amplifying whole human mitochondrial DNA. Phusion DNA Polymerases completed the entire PCR reaction in less than 5 hours, while the other polymerases required almost 8 hours. Compared to the three other polymerases tested, which are recommended by the manufacturers for amplifi cation of long genomic targets, Phusion DNA Polymerases provided high yield with lower enzyme amounts.

An important advantage of Phusion DNA Polymerases is their extremely low error rate. It is 50-fold lower than that of Taq polymerase. Two different buffers are provided with Phusion DNA Polymerases: Phusion HF Buffer (error rate 4.4 x 10^-7) and Phusion GC Buffer (error rate 9.5 x 10^-7). HF Buffer should be used as the default buffer for highfi delity amplifi cation. GC Buffer can, in turn, improve the performance on some diffi cult or long templates. Due to their high accuracy, Phusion DNA Polymerases can reliably be used for studying mitochondrial DNA point mutations. In conclusion, Phusion DNA Polymerases perform accurate and fast amplifi cation of mitochondrial DNA.

Acknowledgements
We thank docent Anu Wartiovaara, Research programme of Neurosciences, University of Helsinki, Finland for the primer sequences.

References
1. Tengan C.H. and Moraes C.T. (1996) Detection and analysis of mitochondrial DNA deletions by whole genome PCR. Biochem Mol Med 58: 130-134.

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Phusion is a trademark of Finnzymes Oy.
PCR license notice: These products are sold under licensing arrangements of Finnzymes Oy with F. Hoffman-La Roche Ltd. The purchase of these products is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front fee, either by payment to Applied Biosystems or as purchased, i.e. an authorized thermal cycler.

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