TABLE 1.
Oligonucleotides used for site-directed mutagenesis
| Mutant or othera | Oligonucleotideb | Oligonucleotide sequence 5′ → 3′c |
|---|---|---|
| L-mutant oligonucleotide | Oli 5′I | CGCGAATTCGATGATAAGCTGTCAAAC |
| Oli 3′D | CGCGAATTCCTCTACTACTTTCCAAGCGTC | |
| L-C1G | MutC94G-VS | CAACTCGAAGTAAACGAAATATT |
| MutC94G-RS | AATATTTCGTTTACTTCGAGTTG | |
| L-U2G | MutU95G-VS | CAACTCGAACGAAACGAAATATT |
| MutU95G-RS | AATATTTCGTTTCGTTCGAGTTG | |
| L-A3C | MutA96C-VS | CAACTCGAACTCAACGAAATATT |
| MutA96C-RS | AATATTTCGTTGAGTTCGAGTTG | |
| L-A4C | MutA97C-VS | CAACTCGAACTACACGAAATATT |
| MutA97C-RS | AATATTTCGTGTAGTTCGAGTTG | |
| L-A5C | MutA98C-VS | CAACTCGAACTAACCGAAATATT |
| MutA98C-RS | AATATTTCGGTTAGTTCGAGTTG | |
| L-C6G | MutC99G-VS | CAACTCGAACTAAAGGAAATATT |
| MutC99G-RS | AATATTTCCTTTAGTTCGAGTTG | |
| L-C1U | MutC94U-VS | CAACTCGAATTAAACGAAATATT |
| MutC94U-RS | AATATTTCGTTTAATTCGAGTTG | |
| L-A3U | MutA96U-VS | CAACTCGAACTTAACGAAATATT |
| MutA96U-RS | AATATTTCGTTAAGTTCGAGTTG | |
| L-A4U | MutA97U-VS | CAACTCGAACTATACGAAATATT |
| MutA97U-RS | AATATTTCGTATAGTTCGAGTTG | |
| L-A5U | MutA98U-VS | CAACTCGAACTAATCGAAATATT |
| MutA98U-RS | AATATTTCGATTAGTTCGAGTTG | |
| L-C6U | MutC99U-VS | CAACTCGAACTAAATGAAATATT |
| MutC99U-RS | AATATTTCATTTAGTTCGAGTTG | |
| B-mutant oligonucleotide | S-3839-VS | GTTGCAACTAGTTCTGACT |
| 3a-169-RS | CAATAATGGAGAGACCAAG | |
| B-C1G | MutC24798G-VS | TTTAAGAAGTAAACTTACGAGTC |
| MutC24798G-RS | GACTCGTAAGTTTACTTCTTAAA | |
| B-U2G | MutU24799G-VS | TTTAAGAACGAAACTTACGAGTC |
| MutU24799G-RS | GACTCGTAAGTTTCGTTCTTAAA | |
| B-A3C | MutA24800C-VS | TTTAAGAACTCAACTTACGAGTC |
| MutA24800C-RS | GACTCGTAAGTTGAGTTCTTAAA | |
| B-A4C | MutA24801C-VS | TTTAAGAACTACACTTACGAGTC |
| MutA24801C-RS | GACTCGTAAGTGTAGTTCTTAAA | |
| B-A5C | MutA24802C-VS | TTTAAGAACTAACCTTACGAGTC |
| MutA24802C-RS | GACTCGTAAGGTTAGTTCTTAAA | |
| B-C6G | MutC24803G-VS | TTTAAGAACTAAAGTTACGAGTC |
| MutC24803G-RS | GACTCGTAACTTTAGTTCTTAAA |
a
Virus names were derived from leader CS mutants (L) or CS-3a mutants (B) and indicate the nucleotide substitution and its position in the CS.
b
Oligonucleotides including the punctual mutations are named “Mut” and indicate the nucleotide substitution and its position at the TGEV genome. VS, virus sense; RS, reverse sense.
c
The mutated nucleotide is shown in boldface. Restriction endonuclease sites used for cloning are shown in italics (EcoRI, GAATTC; SpeI, ACTAGT). CS and cCS are underlined.