Table 1. SDM using mutagenic primers of different length, GC content, and Tm.
| K60A2 | L100A2 | K80A2 | F200A2 | Q97H1 | D107A1 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Lengtha | 45* | 29* | 45 | 29 | 45 | 29* | 45* | 29 | 37* (QCM) | 29* | 41* (QCM) | 29* | |||
| % GC | 33 | 34 | 42 | 41 | 42 | 45 | 38 | 38 | 43 | 41 | 41 | 48 | |||
| Tmb | 76 | 65 | 80 | 68 | 80 | 70 | 78 | 67 | 78 | 71 | 79 | 74 | |||
| Coloniesc | exp 1 | 5 | 40 | 38 | 118 | exp 3 | 3 | 25 | 17 | 5 | exp 5 | 43 | 36 | 6 | 53 |
| exp 2 | 1 | 40 | 14 | 166 | exp 4 | 2 | 16 | 1 | 10 | exp 6 | 61 | 63 | 1 | 78 | |
| Mutation frequencyd | exp 1 | 2/3 | 2/3 | 2/3 | 2/3 | exp 3 | 3/3 | 1/3 | 3/3 | 2/3 | exp 5 | 16/16 | 15/16 | 4/6 | 16/16 |
| exp 2 | 0/1 | 3/3 | 3/3 | 3/3 | exp 4 | 2/3 | 2/3 | 1/1 | 3/3 | exp 6 | 16/16 | 16/16 | 1/1 | 16/16 | |
The superscripts in the distinct mutations indicate the number of nucleotide mismatches of each mutation with respect to the wild type sequence. pRK5-PTEN served as template plasmid.
*One of the primers from the mutagenic primer pair has G or C in 3’ position.
aPre-defined length primers follow the design n+3+n (underlined is the mutated codon; 45-mer, 21+3+21; 29-mer, 13+3+13). QCM web-based primers were: Q97H, 13+3+16; D107A, 19+3+19.
bTm was calculated according to the QuikChangeTM manual (Agilent Technologies).
cNumber of bacteria colonies after transformation of the DpnI-digested PCR product; exp 1 to 6 are independent experiments.
dNumber of colonies with mutation/number of colonies analyzed. Mutations were identified by DNA sequencing or, in the case of Q97H and D107A mutations, by DNA sequencing or restriction analysis.