PCR-based strategies that were used for the construction of mutant minigenomes.

(A) Inside-out primer deletion mutagenesis. (B) Overhang/inside-out primer insertion mutagenesis. (C) Complementary primer mutagenesis for introduction of insertions or substitutions. In approaches A and B the entire plasmid is amplified with 5‘-phosphorylated primers that introduce the desired insertions/deletions, followed by circularization of PCR products and template removal by Dpn I treatment before bacterial transformation. In approach C phosphorylation of 5’-ends and ligation are carried out by bacterial enzymes after DNA transformation.

(DOCX)