The Power of Two—Fusion DNA polymerases

The idea of creating fusion DNA polymerases was inspired by the natural cellular replication machinery. Replication systems in cells rely on highly processive DNA polymerases that can incorporate thousands of nucleotides without dissociating from the DNA template. Their high processivity is achieved through interaction with “processivity-enhancing proteins” by:

• Binding directly to dsDNA to increase polymerase-DNA binding efficiency, and/or
• Preventing the polymerases from dissociating from their DNA templates.

Traditional DNA polymerases used in PCR commonly have low processivity and incorporate only a few nucleotides per binding event. This is because they often lack the polymerase processivity factors, resulting in:  

• Requiring one minute or more to amplify 1 kb of DNA template,
• Being unable to amplify amplicons longer than 4–5 kb,
• Being affected by inhibitors such as ethanol, EDTA, or other factors in DNA samples.

Low processivity of PCR enzymes can be a limiting factor for PCR efficiency. Therefore, increasing DNA polymerase processivity is important for improving PCR performance and helping enable new applications.

Protein engineering based on fusion technology was employed to improve performance of DNA polymerases in PCR [1]. Mimicking natural replication machinery, a dsDNA-binding protein was fused to a DNA polymerase as its processivity-enhancing domain. The dsDNA-binding protein of choice was Sso7, derived from the thermophilic archea, Sulfolobus sulfactaricus.

Sso7 is a 7 kDa nonspecific DNA–binding protein that naturally functions in chromatin remodeling. When Sso7 was fused to low-processive DNA polymerases, the polymerases became highly processive, and also demonstrated other benefits such as higher efficiency, faster DNA synthesis, and longer amplification (e.g., 15 kb fragment).

Polymerases with Sso7 displayed high tolerance to PCR inhibitors from tissues and DNA samples. This property has further enabled practical applications such as Direct PCR, which allows DNA amplification directly from tissue samples without the need for DNA purification or extraction.

One of the best known and widely used fusion PCR enzymes is the family of Thermo Scientific Phusion High-Fidelity DNA Polymerases. Phusion DNA polymerase was created by fusing the Pyrococcus-like proofreading DNA polymerase to Sso7. This combination resulted in a high-fidelity enzyme (>100x that of Taq DNA polymerase, for Phusion Plus DNA Polymerase) with high processivity that is capable of amplifying templates up to 20 kb, short reaction time (15 sec/kb), and inhibitor tolerance. With its introduction in 2003, Phusion DNA polymerase established a new standard for high-performing PCR. Phusion products have been referenced in thousands of publications and have become a DNA polymerase of choice for applications ranging from reconstruction [2], design [3] and massively-parallel, high-throughput sequencing of whole genomes [4].

Using similar fusion technology, higher-performing proofreading DNA polymerases have been developed, such as Invitrogen Platinum SuperFi II DNA Polymerase. Platinum SuperFi II DNA Polymerase offers ultrahigh fidelity (>300x that of Taq DNA polymerase), higher tolerance to PCR inhibitors, and enhanced amplification of long and/or challenging templates.

References

1. Wang Y, Prosen DE, Mei L et al. (2004) A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 32(3):1197-1207.
2. Gibson DG, Benders GA, Andrews-Pfannkoch C, et al. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319(5867):1215-1220.
3. Gibson DG, Glass JI, Lartigue C, et al. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329 (5987):52-56.
4. Kinde I, Wu J, Papadopoulos N, Kinzler KW et al. (2011) Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A. 108(23):9530–9535.