Table 1.
Oligonucleotides used for the amplification, cloning and quantification of partial DENV genome fragments.
| Oligonucleotide name | Orientation | Restriction endonucleases | Amplicon name | Sequence (5′ → 3′)a, b |
|---|---|---|---|---|
| CMV5 | F | Sfo I | CMV, CMV-DEN2 5′ | aatgatggcgccttgacattgattattgactagttattaatag |
| CMV3 | R | CMV | ggtccacgtagactaacaactacggttcactaaacgagctctgc | |
| DEN2L5 | F | DEN2 5′ | agttgttagtctacgtggaccgacaaagacag | |
| DEN2L3 | R | Hind III; Pml I; Nsi I; Sph I | DEN2 5′, CMV-DEN2 5′ | cgcgaagctttggccacgtgcaggatgcatgactgcatgctcttcccctgagtgaggtg |
| DEN2R5 | F | Pml I | DEN2 3′, DEN2 3′-RZ-BGH | gaagtcggcacgtgaggctgttgaagatagt |
| 3UTR-RZ-R | R | DEN2 3′ | atgccgacccagaacctgttgattcaacagcaccatt | |
| RZ-F | F | RZ-BGH | aacaggttctgggtcggcatggcatctccacctcctcg | |
| BGH3 | R | Hind III; Sac II | RZ-BGH, DEN2 3′-RZ-BGH | gccctaagcttccgcggtcgagctctccccaAcatgcctgctattgtcc |
| DENV1385-4701F | F | Sfo I | 1385-4701, 1385-ENV | aatgatggcgccggaatacaccattgtgataac |
| DENV1385-4701R | R | Sph I | 1385-4701, NS1-4701 | gaagagcatgctggttcaatcctctttcctttat |
| DENV4702-8834F | F | Sph I | 4702-8834 | gaagagcatgcccagatcggagccggagtttacaa |
| DENV4702-8834R | R | Sph I | 4702-8834 | gaagagcatgcaccagctcccaaaacctactatct |
| NS1-F | F | Sfo I; Mlu I | NS1-4701 | aatgatggcgccgcgcacgcgtacgctgtatttgggagttat |
| ENV-R | R | Mlu I | 1385-ENV | gcgcacgcgtggcctgcaccataactcccaaat |
| PEGFP-F | F | Mlu I | EGFP | gcgcacgcgtatggtgagcaagggcgaggag |
| PEGFP-R | R | Mlu I | EGFP | gcgcacgcgtcttgtacagctcgtccat |
| hRLuc-F | F | Mlu I | hRLuc | gcgcacgcgtatggcttccaaggtgta |
| hRLuc-R | R | Mlu I | hRLuc | gcgcacgcgttaactgctcgttcttcag |
| NS5_F | F | NS5 | tcacaccatttccatgagttaatca | |
| NS5_R | R | NS5 | cgggctctaccaatcagttca | |
| NS5_DENV (probe) | F | FAM-ccgcgtacttgtagttccatgcagaaacc |
a
The underlined sequences correspond to recognition sites for the restriction endonucleases used in the cloning strategy.
b
The boldface sequences correspond to oligonucleotide regions that hybridize with the DENV genome.
c
The uppercase letter in the BGH3 sequence corresponds to a punctual mutation introduced to eliminate the Sph I restriction site. F: forward; R: reverse; FAM: 6-carboxyfluorescein.