Table 6. Iterative one-tube-only SDM using large combinations of primers targeting the same residue.
| Mutations targeteda | Colonies | Colonies analyzed | Mutations obtainedb |
|---|---|---|---|
| PTEN A126 1st | |||
| C2-E2-F3-H2-I2-K3-L2-N2-P1-Q3 R2-T1-W3-Y3 | 194 | 14 | 0-0-0-4-0-1-0-1-0-1 1-2-1-1-2 |
| PTEN A126 2nd | |||
| D1-E2-F3-G1-I2-L2-M3-P1-S1-V1 | 106 | 10 | 1-0-1-2-1-1-0-0-2-1-1 |
| PTEN G129 1st | |||
| C3-F3-H3-I2-K2-L2-M3-N3-P2-Q2 S1-T2-W2-Y3 | 24 | 14 | 0-0-1-5-0-0-0-0-0-0 0-2-4-0-2 |
| PTEN G129 2nd | |||
| A1-D2-E1-F3-K2-L2-M3-N3-P2-Q2 R1-S1-V1-Y3 | 68 | 14 | 2-1-3-0-0-0-1-0-0-2 1-0-2-1-1 |
| PTPRZ-B L1454 1st | |||
| A2-C2-D3-E2-F1-G2-H3-I1-K2-M2 N2-P2-Q2-R2-S1-T2-V1-W2-Y2 | 264 | 20 | 1-0-1-0-3-0-2-0-1-0 0-1-3-1-2-1-1-1-1-1 |
| PTPRZ-B L1454 2nd | |||
| C2-E2-G2-I1-M2-N2 | 11 | 7 | 0-2-0-3-2-0-0 |
Data and mutations obtained are represented as in Table 5. pRK5-PTEN or pCDNA3-PTPRZ-B served as template plasmids.
aTwo iterative PCR mutagenic reactions (1st and 2nd) were sequentially run for each targeted residue (A126, G129, and L1454). The mutations targeted in each case are indicated with the amino acid one-letter code, and the superscripts indicate the number of nucleotide mismatches of each mutation with respect to the wild type sequence.
bNumber of samples obtained for each mutation are underlined and indicated following the order of the mutations targeted, and last number (not underlined) corresponds to wild type.