Table 4. One-tube-only SDM using combinations of primers targeting consecutive residues.
| Mutations targeteda | Colonies | Colonies analyzed | Mutations obtainedb,c |
|---|---|---|---|
| I502-D511 | 63 | 4 | 0-3-1 |
| V541-R552 | 30 | 4 | 4-0-0 |
| D581-S591 | 38 | 4 | 1-1-2 |
| K602-H612 | 39 | 4 | 3-1-0 |
| H642-Y652 | 166 | 4 | 0-4-0 |
| Y682-N692 | 120 | 4 | 4-0-0 |
| I502-D511-D521 | 41 | 10 | 3-7-0-0 |
| K602-H612-K622 | 86 | 10 | 8-0-0-2 |
| N632-H642-Y652 | 82 | 10 | 0-3-5-2 |
| I502-D511-D521-V531 | 28 | 14 | 0-7-2-4-1 |
| V541-R552-F562-L572 | 35 | 14 | 4-4-3-1-2 |
| K602-H612-K622-N632 | 52 | 14 | 6-1-6-0-1 |
| H642-Y652-K662-I672 | 32 | 14 | 2-4-3-2-3 |
| I502-D511-D521-V531-V541-R552 | 19 | 16 |
0-0-1-1-0-0-0 I50A+V54A (2) I50A+R55A (12) |
| K602-H612-K622-N632-H64 -Y652 | 98 | 16 |
3-1-4-0-0-0-2 K60A+Y65A (6) |
| I502-D511-D521-V531-V541-R552-F562-L572 | 62 | 16 |
0-0-0-0-0-0-0-0-3 I50A+R55A (1) D51A+L57A (9) D52A+L57A (3) |
| K602-H612-K622-N632-H642-Y652-K662-I672 | 33 | 16 |
0-0-0-0-0-2-0-0-3 K60A+Y65A (1) K60A+K66A (3) K60A+I67A (2) H61A+K66A (1) H61A+I67A (3) K62A+I67A (1) |
Data are represented as in Table 1. pRK5-PTEN served as template plasmid.
aAll mutations are Ala substitutions (I50A, etc), and the superscripts indicate the number of nucleotide mismatches of each mutation.
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 (for instance, in first row: I50A, 0 samples; D51A, 3 samples; wild type, 1 sample).
cThe double mutations obtained are indicated, and the number of samples in each case are in brackets (for instance, in 14th row: double mutation I50A+V54A, 2 samples).