Uracil Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA-fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming.
Here we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (Tm), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analyzed in terms of GC content, presence of GC clamp at 3'-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures), and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options.
Lars Rønn Olsen, Niels Bjørn Hansen, Mads Tvillinggaard Bonde, Hans Jasper Genee, Dorte Koefoed Holm, Simon Carlsen, Bjarne Gram Hansen, Kiran Raosaheb Patil, Uffe Mortensen, Rasmus Wernersson
PHUSER (Primer Help for USER):
A novel tool for USER fusion primer design
Nucl. Acids Res. 2011, submitted
Rasmus Wernersson: firstname.lastname@example.org