Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR

Abstract

AbstractThermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, other properties than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA and that is unaffected by replacing dTTP with dUTP in PCR. This makes it an attractive DNA polymerase for use e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Furthermore, Neq2X7 is easy to produce, and the expression plasmid is freely available.Graphical abstract