Advancing USER cloning into simpleUSER and nicking cloning.

The two novel methods for DNA cloning presented here have been developed for the rapid construction of vectors used for insertion of genes in filamentous fungi. The current study shows that both simpleUSER cloning and nicking cloning can substitute USER cloning for insertion of single PCR fragments into plasmids. The simpleUSER cloning method proposed in this paper varies from USER cloning by substituting the dual enzymatic plasmid preparation step with a single enzymatic step. The other method further abolishes the use of USER™ enzyme mix and PfuTurbo Cx polymerase, and is referred to as nicking cloning. We show that both simpleUSER cloning and nicking cloning can substitute USER cloning for insertion of single PCR fragments into plasmids, and that the combination of these two methods works efficiently for the construction of selective plasmids and plasmids for co-transformation. This strategy was applied to genetically modify the filamentous fungus Aspergillus carbonarius. The two methods simplify DNA cloning by reducing time and complexity associated with cloning in filamentous fungi.