TABLE 1.
Oligonucleotide primers used in this worka
| Primer no. | Primer | Sequence | Characteristics and use |
|---|---|---|---|
| 1 | ORF1pMalfor | 5′-GAAGATCTATGAA-GTTTCCTGTTCCG-3′ | 5′ primer for DNA fragment containing orf1 used for pMal-c2X vector |
| 2 | ORF1polyTfor | 5′-ATGAAGTTTCCTGTTCCGCAC-3′ | 5′ primer for DNA fragment containing orf1 used for pETBlue-1 vector |
| 3 | ORF1SalIrev | 5′-CCATGTCGACCACTCCCTCAATCC-3′ | 3′ primer for DNA fragment containing orf1 used for pETBlue-1 and pMal-c2X vectors |
| 4 | ORF2BglIIfor | 5′-GAGGGAAGATCTGACATGGGAAG-3′ | 5′ primer for DNA fragment containing orf2 used for pMal-c2X vector |
| 5 | ORF2polyTfor | 5′-ATGGGAAGTATCGTTTCCAC-3′ | 5′ primer for DNA fragment containing orf2 used for pETBlue-1 vector |
| 6 | PEPSSalIrev | 5′-GATCGGATTGGTCGACATCCA-3′ | 3′ primer for DNA fragment containing orf2 used for pETBlue-1 and pMal-c2X vectors |
| 7 | ORF3polyTfor | 5′-ATGATCGTACGCAACTGGATG-3′ | 5′ primer for DNA fragment containing orf3 used for pETBlue-1 vector |
| 8 | ORF3polyTrev | 5′-GACCTCGGTGGTGATTCTCTT-3′ | 3′ primer for DNA fragment containing orf3 used for pETBlue-1 vector |
| 9 | 5′orf1for | 5′-CACCACGGGTCGTATTGA-3′ | RT-PCR, negative control |
| 10 | orf1Cy5rev | 5′-CTCCGGGTAATGCAATCCGTCG-3′ | RT-PCR, negative control and primer extension experiment |
| 11 | orf1for | 5′-GCTCTGGGGGGTCACCA-3′ | RT-PCR, amplification of intergenic region between ORFs 1 and 2 |
| 12 | orf2rev | 5′-CGCACTTGCCGCCCACCA-3′ | RT-PCR, amplification of intergenic region between ORFs 1 and 2 |
| 13 | orf2for | 5′-GACAACTTCGTCGTCAACAAG-3′ | RT-PCR, amplification of intergenic region between ORFs 2 and 3 |
| 14 | orf3rev | 5′-GTGGATATTGGCTTCGGAAA-3′ | RT-PCR, amplification of intergenic region between ORFs 2 and 3 |
| 15 | orf3for | 5′-TGTTCTCGCTGGCGGCGCAT-3′ | RT-PCR, amplification of intergenic region between ORFs 3 and 4 |
| 16 | orf4rev | 5′-AAGAGAATCACCACCGAGGTC-3′ | RT-PCR, amplification of intergenic region between ORFs 3 and 4 |
| 17 | orf4for | 5′-CCTACGAGTGGAAGCAGAAGC-3′ | RT-PCR, amplification of intergenic region between ORFs 4 and 5 |
| 18 | orf5rev | 5′-TGTTCATCTCCTCGAGCA-3′ | RT-PCR, amplification of intergenic region between ORFs 4 and 5 |
| 19 | orf5for | 5′-CGGCATGGGCGTGATCGTGCTGG-3′ | RT-PCR, amplification of intergenic region between ORFs 5 and 6 |
| 20 | orf6rev | 5′-CCCAGTCCACTTCCTGTCTGAT-3′ | RT-PCR, amplification of intergenic region between ORFs 5 and 6 |
| 21 | orf6for | 5′-GGAGCGCGGCATCCACGTGTTC-3′ | RT-PCR, amplification of intergenic region between ORFs 6 and 12 |
| 22 | orf12rev | 5′-GCCGCGGGCGGACAAAAGGC-3′ | RT-PCR, amplification of intergenic region between ORFs 6 and 12 |
| 23 | orf12for | 5′-CCGCATCATCGAGACCGTCCA-3′ | RT-PCR, amplification of intergenic region between ORFs 7 and 12 |
| 24 | orf7rev | 5′-AAGGAAGTGATCCACCGCC-3′ | RT-PCR, amplification of intergenic region between ORFs 7 and 12 |
| 25 | orf7for | 5′-GGCACATCGAAAGTCGGGTT-3′ | RT-PCR; amplification of intergenic region between ORFs 7 and 8 |
| 26 | orf8rev | 5′-TGGTGATGTCGGATTCGG-3′ | RT-PCR, amplification of intergenic region between ORFs 7 and 8 |
| 27 | orf8for | 5′-GGCATGATCATCGCGCCCTGT T-3′ | RT-PCR, amplification of intergenic region between ORFs 8 and 9 |
| 28 | orf9rev | 5′-GATGCCACCGATCGCCCTTTCC-3′ | RT-PCR, amplification of intergenic region between ORFs 8 and 9 |
| 29 | orf9for | 5′-GGGATACAGCATCCGCAT-3′ | RT-PCR, amplification of intergenic region between ORFs 9 and 10 |
| 30 | orf10rev | 5′-GCCATGTGAATCTCCTTGCTGACG-3′ | RT-PCR, amplification of intergenic region between ORFs 9 and 10 |
| 31 | orf10for | 5′-GCATACCGATCGAACACTGG-3′ | RT-PCR, amplification of intergenic region between ORFs 10 and 13 |
| 32 | orf13rev | 5′-CGTTCCCTTGACGAGGAAGG-3′ | RT-PCR, amplification of intergenic region between ORFs 10 and 13 |
| 33 | orf13for | 5′-GGCGCTGACGAAGATGGAA-3′ | RT-PCR, amplification of intergenic region between ORFs 13 and 14 |
| 34 | orf14rev | 5′-TCCACTTGTTGGTGTAGAAGC-3′ | RT-PCR, amplification of intergenic region between ORFs 13 and 14 |
| 35 | orf14for | 5′-GAGTTCCGCATCAAGGTCAG-3′ | RT-PCR, amplification of intergenic region between ORFs 14 and 15 |
| 36 | orf15rev | 5′-CTCCGTTCGGATACTGGTCG-3′ | RT-PCR, amplification of intergenic region between ORFs 14 and 15 |
| 37 | orf15for | 5′-CATTGCGCAATGGCAGCACG-3′ | RT-PCR, amplification of intergenic region between ORFs 15 and X |
| 38 | orfXrev | 5′-CTTGAGCCGCTCCTTGCTCG-3′ | RT-PCR, amplification of intergenic region between orfs 15 and X |
a
The mutated nucleotides are marked in italic type, and the restriction sites are marked in boldface type. For gene order, see Fig. 2A.