TABLE 1.
Oligonucleotides used in this study
| Designation | Sequence, 5′-3′a | Use |
|---|---|---|
| 898 | TGAGTAAAGCTTTTATTGCAGAAAGCCATCCCG | Amplify rhaS, downstream |
| 1170 | CCGGAATTCTTGTGGTGATGTGATGCTCAC | Amplify rhaS, upstream |
| 2064 | TCTCTTTCACTGGCTACGCTACATCG | Arg202Ala |
| 2065 | CGTACGCTACATGCGCAGCTTAAGCAG | Arg206Ala |
| 2066 | CAATTTTCTCTTTCAGCGCGTACGCTACAT | Leu201Ala |
| 2067 | CCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTCGACGCTTTb | H-T-H 2 random mutations |
| 2068 | ATGACCGTATTACATAGTGTGGATc | rhaS sequencing |
| 2069 | TTATTGCAGAAAGCCATCCCGTCCc | rhaS sequencing |
| 2073 | GGATTCAGCGCCAGTAACCAC | Asp250Ala |
| 2074 | TGGTTGCACAGATGGAACAGCc | rhaS sequencing |
| 2075 | GTTGAGACGTGATGCGCTGTTc | rhaS sequencing |
| 2087 | TTCAGCGACAGTGCCCACTTT | Asn252Ala |
| 2088 | TGTGGAGCCAGCGACAGTAAC | Phe248Ala |
| 2089 | AGTAACGCCTTTTCGACGCTTTTT | His253Ala |
| 2090 | AACCACGCTTCGACGCTTTTT | Phe254Ala |
| 2091 | AGTAACCACTTTGCGACGCTTT | Ser255Ala |
| 2093 | TATCGCGCTGGATTCAGC | Cys246Ala |
| 2120 | CGTACGCTAGCTCGGCAGCTTA | His205Ala |
| 2125 | CCACTTTTCGGCGCTTTTTCG | Thr256Ala |
a
For oligonucleotides used for PCR amplification, sequences complementary to rhaS are underlined; for those used for site-directed mutagenesis, positions of mutations are underlined.
b
During synthesis, the wild-type nucleotide as well as a low concentration of each of the other three nucleotides was added at each of the underlined positions, resulting in a pool of oligonucleotides with an average of approximately 1.5 mutations each.
c
IRD41 dye labeled for use in a LI-COR automated sequencer.