Fig 1.
Amino acid substitution mutagenesis using mutagenic primers of different length, Tm, and GC composition.
(A) Mutagenic primer pairs of pre-defined length (45-mer or 29-mer) or as suggested by the web-based QuikChange Primer Design Program (QCM primers) were used to create various PTEN mutations (pRK5-PTEN as template plasmid). Features of the mutagenic primers are listed in Table 1. Upper images show 10 μl of the respective PCR product, or of BstEII-digested λ phage as size marker (kb, lanes 1), following electrophoresis on 1% agarose gels. Lower panels display 1% agarose gel electrophoresis results for purified plasmid DNA (~3 μg) after restriction enzyme digestion. To reveal the 1.2 kbp PTEN insert (mutations K60, L100A, K80A, and F200A) an XbaI/SalI double digestion was used and one sample per SDM reaction is shown. To monitor the mutagenesis efficiency (mutations Q97H and D107A) we used XbaI/SalI or BglII, respectively, and three samples are shown per SDM reaction. Digested wild type pRK5-PTEN (wt, lanes 2) was included as a control. Gels correspond to experiments 1, 3, and 5 listed in Table 1. (B) PCR results using mutagenic (29-nucleotides) primer pairs generating 11 different Lys-to-Arg amino acid substitutions at different PTEN regions (upper panel; pRK5-PTEN as template plasmid), or generating 12 different amino acid substitutions at the intracellular region of PTPRZ-B (lower panel; pENTR-PTPRZ-B as template plasmid). Again, 10 μl of the PCR product was resolved on 1% agarose gels. Corresponding experiments and primer features are listed in Table 2. Molecular sizes of the linearized plasmids (6 and 7.3 kb) or the restriction enzyme-generated DNA fragments, are indicated left of the images in panels A and B.
Table 1.
SDM using mutagenic primers of different length, GC content, and Tm.
Table 2.
Standardized SDM using mutagenic primers of 29-mer length and different GC content and Tm.
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”“.
Table 3.
One-tube-only SDM using combinations of template plasmids.
Table 4.
One-tube-only SDM using combinations of primers targeting consecutive residues.
Fig 3.
Data plots representing the obtaining (extraction) of all different individual mutations (samples to be extracted) from a given mix of mutations, assuming a stochastic distribution.
The x axis indicates the number of different mutations included in the one-tube-only mutagenesis reaction, and the y axis indicates the number of samples (bacteria colonies) to be analyzed to obtain at least one of each mutation with a certain probability (95%, in red; 90%, in blue; 85%, in green). Dots indicate data obtained from a computer-assisted simulation, up to a mix of 15 different mutations. Solid lines indicate the probability distribution plots (Theory) up to a mix of 6 different mutations. Numeric values (non-brackets, simulation; brackets, theory) are indicated in the inserted table up to a mix of 6 different mutations.
Table 5.
One-tube-only SDM using combinations of primers targeting the same residue.
Table 6.
Iterative one-tube-only SDM using large combinations of primers targeting the same residue.