A Decade of Innovation in DNA Polymerase Technology
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The Thermo Scientific Phusion High-Fidelity DNA Polymerases are currently celebrating their 10 year anniversary. I used this opportunity to catch up with Thermo Fisher Scientific to learn more about these high-fidelity DNA polymerases.
AB: For our readers that may not have heard of the Phusion name, can you tell me more about this range?
Thermo Fisher: Ten years ago a team in Finland succeeded in developing a very robust DNA polymerase that had an error rate that was 50 fold lower than that of conventional Taq polymerase. The enzyme, which is based on unique fusion protein technology, was developed in an effort to improve PCR efficiency and to find a solution to several PCR limitations. The fusion protein technology is made up of two constituent parts fused together, a processivity factor (a thermophilic, non-specific, double-stranded DNA binding protein) and a proofreading DNA polymerase. In addition to the expected increase in reliable read length, this PCR enzyme exhibits a number of other desirable features, including increased fidelity (52X over Taq DNA polymerase), resistance to various PCR inhibitors and faster overall performance. The enzyme has enabled the streamlining of many PCR applications and is still leading the High Fidelity PCR market today.
Over the last ten years the Phusion product family has grown to include stand-alone enzymes, master mixes and Hot Start enzymes, all with a visible green buffer that enables the direct loading of reaction products onto gels. These fusion enzymes enabled the creation of a direct PCR protocol, where users do not purify DNA at all; this means that samples (plant, animal tissue or blood) can be placed directly into a PCR mix for amplification.
AB: What key features does Phusion offer researchers?
Thermo Fisher:
• Extremely high fidelity – 52X times higher than commonly used Taq DNA polymerases. This allows researcher’s to generate PCR products with a very low chance of sequence errors.
• Long amplicons – higher processivity enzyme enables the generation of products up to 20 kb.
• Fast amplification – allows users to dramatically reduce PCR cycling times
• Resistance to inhibitors – allows users to even amplify templates of suboptimal purity
AB: Has Phusion enabled any notable research?
Thermo Fisher: Phusion is designed to streamline the amplification process, reduce the number of sequence errors and work effectively with challenging templates. Phusion is often utilised at the optimization and advancement stages of protocols, rather than at the beginning of new method development. However, there are a vast number of groups that have used Phusion to complete their initial projects, as they were unable to achieve the necessary level of technical sensitivity with standard polymerases. In fact, we were delighted to see that our high fidelity Phusion enzyme enabled the development of a technique called BEAMing by a team at Inostics. This technique is unique in that it allows the sensitive detection of rare mutations in ctDNA and facilitates cancer research and diagnosis.
AB: You recently expanded the range with the launch of Phusion Green High-Fidelity DNA Polymerase, how does this product complement the existing range?
Thermo Fisher: Increasingly our customers are looking to make time, materials and cost savings during every step of their workflows. In an effort to help researchers achieve these savings, we provide our Phusion products with a buffer solution that contains electrophoresis tracking dyes and density reagents. This enables our customers to remove a number of steps from their workflows, such as the need to purchase dyes and their subsequent use in pipetting steps before running gels. Put simply, the PCR product can be loaded directly onto a gel after cycling. Our customers love that.
AB: Finally can you tell me a little more about what the future holds for Phusion?
Thermo Fisher: Phusion DNA polymerases, as with all other proofreading enzymes, are not tolerant to dUTP, an unnatural nucleotide present in DNA after certain procedures like bisulphite conversion. Therefore some of our customers will benefit from Phusion U, a new mutant version of Phusion that is due to be launched soon. Phusion U will be able to tolerate dUTP in DNA templates, as well as being able to incorporate dUTP for techniques such as carry over contamination using UDG.
Even without additional enhancements, new customers are continuing to employ Phusion High-Fidelity DNA polymerases in their new applications. We believe that our Phusion technology will be in demand for many years to come.
More information on Phusion High-Fidelity DNA Polymerases and the Phusion Fest can be found by visiting www.thermoscientific.com/phusion
Thermo Fisher were speaking to Ashley Board, Managing Editor for Technology Networks. You can find Ashley on Google+ and follow Technology Networks on Twitter.
