Table 1.
Results of mutagenesis experiments using the novel three-primer, one-step PCR protocol
| Mutant name | Original sequences/template (bp) | Mutagenic primer with mutant sequences | Mutagenesis efficiencya (%) |
|---|---|---|---|
| CNB/K134H | AAA/CNB (554) | GCAGATTGTAGACCACACCATAATAAACG | 7/7 = 100 |
| CNB/G120GGb | GGC/CNB (554) | GCGTATCTTTCAGGTTGTTGCCGCCCACCATCATCTTCAACAC | 7/7 = 100 |
| CNB/G120c | GGC/CNB (554) | GAAGATGATGGTGAACAACCTGAAAGATACGCAGTTACAG | 5/5 = 100 |
| CNB/K134R | AAA/CNB (554) | AGATTGTAGACCGCACCATAATAAACGCA | 1/1 = 100 |
| CAN/RY112-3LT | CGCTAC/CNA (1577) | CCCCTAAGAAGAGTGTCAAAGTGTTGGCAGGAGAT | 1/1 = 100 |
| CAN/V314H | GTG/CNA (1577) | GCACCAAATTACTTAGATCATTACAATAATAAAGC | 1/1 = 100 |
| PP1/L88L | CTG/PP1 (1034) | CACGTAGTCACCCAGGAATAGGTAGTTGCTCTC | 1/1 = 100 |
| CNB/M118N | ATG/CNB (554) | TTGAAGATGAATGTGGGCAACAACCTGAAAGATACGC | 2/2 = 100 |
| CNB/M118K | ATG/CNB (554) | TGAAGATGAAAGTGGGCAACAAC | 1/1 = 100 |
| CNB/M118W | ATG/CNB (554) | TTGAAGATGTGGGTGGGCAACAACCTGAAAGATACG | 1/1 = 100 |
| CNB/M118G | ATG/CNB (554) | TTGAAGATGGGCGTGGGCAACAACCTGAAAGATAC | 1/1 = 100 |
| CNB/M118H | ATG/CNB (554) | TTGAAGATGCATGTGGGCAACAACCTGAAAGATACG | 1/1 = 100 |
| CNB/M118E | ATG/CNB (554) | TTGAAGATGGAAGTGGGCAACAACCTGAAAGATACGC | 1/1 = 100 |
| CNB/V119R | GTG/CNB (554) | TTGAAGATGATGCGTGGCAACAACCTGAAAGATA | 1/1 = 100 |
| CNB/G120Y | GGC/CNB (554) | TTGAAGATGATGGTGTATAACAACCTGAAAG | 1/1 = 100 |
| CNB/G120R | GGC/CNB (554) | TTGAAGATGATGGTGCGTAACAACCTGAAAG | 1/1 = 100 |
| CNB/L123R | CTG/CNB (554) | ATGGTGGGCAACAACCGCAAAGATACGCAGT | 1/1 = 100 |
aMutagenesis efficiency was calculated as the number of confirmed mutants out of a number of randomly selected clones.
bCNB/G120GG represents bases insertions.
cCNB/G120 represents bases deletions.