Fig 2. Schematic depiction of the different one-tube-only SDM approaches used in this study.

The cDNAs are represented as lines divided in 3-mer base codons. (A) Strategy for the simultaneous introduction of a mutation (in red) in several background plasmids by a mixed templates SDM reaction. Wild type residue is indicated as 1, and mutated residue as 1’. The 13+3+13 design of our standardized mutagenic primer pairs is indicated. Plasmids are indicated as A and B. (B) Strategy for one-tube parallel substitution of sequential amino acids (scanning mutagenesis) by mixing overlapping mutagenic primers targeting adjacent residues in the SDM reaction. Wild type residues are indicated as 1 to 8, and mutated residues (in red and blue) as 1’ to 8’. Note that individual mutations are obtained when primer pair overlap is more than five codons, whereas multiple mutations are obtained when the overlap is smaller. (C) Strategy for the simultaneous substitution of one residue to a collection of distinct residues (single site-multiple mutagenesis) by mixing distinct mutagenic primers targeting the same residue in the SDM reaction. Wild type residue is indicated as 1, and mutated residues (in red) as 1’ to 1”“.