TABLE 1.
Summary of oligonucleotides used for RT-PCR amplification
| Primera | Sequence (5′ → 3′)b | Position within VR-2332 genomec |
|---|---|---|
| 5′ terminus (fragment b) primers | ||
| RT416 | 5′ TGC GAT TGG A 3′ | 435-445 |
| F20 | 5′ CTC GAG GGC GCG CCT AAT ACG ACT CAC TAT AGG TAT GAC GTA TAG GTG TTG GCT 3′ (Xho1, AscI) | 1-21 |
| R229 | 5′ CGT GTG CAG TAT ACT TGG CCC T 3′ (BstZ17I) | 249-270 |
| Fragment e primers | ||
| RT4890 | 5′ AAG GCT TGG A 3′ | 4910-4919 |
| F251 | 5′ GGA GGG CCA AGTATA CTG CAC ACG A 3′ (BstZ17I) | 247-271 |
| R4774 | 5′ GTG TCA GGG TCA ACC ACG A 3′ | 4794-4812 |
| Fragment f primers | ||
| RT7924 | 5′ TGC ATC AGC A 3′ | 7944-7953 |
| F4333 | 5′ ATC TTG GCT GGA GCT TAC GT 3′ | 4334-4353 |
| R7821 | 5′ TGG TTG TGC TCA ACC GCG T 3′ | 7841-7859 |
| Fragment d primers | ||
| RT13019 | 5′ AGC TGA AGG A 3′ | 13039-13048 |
| F7406 | 5′ TCT CAG AGT TGG CGA CCC T 3′ | 7408-7426 |
| R12887 | 5′ ATC CTG CAC CAA AGA GAC CT 3′ | 12907-13026 |
| Fragment c primers | ||
| RT15327 | 5′ AAT TGA ATA GGT 3′ | 15347-15358 |
| F12513 | 5′ TTT CAG CAT CTA GCC GCC A 3′ | 12515-12533 |
| R15278 | 5′ AAT CAG TGC CGT TAA CCA CAC ATT CTT CCA 3′ (HpaI) | 15298-15327 |
| 3′ terminus (fragment a) primers | ||
| RT15392 | 5′ CAG GAA ACA GCT ATG ACA CCT GAT CTC TAG A AA CGT T(T)383′ (XbaI, AclI) | Poly (A) tail |
| F15301 | 5′ AGA ATG TGT GGTTAACGG CA 3′ (HpaI) | 15302-15321 |
| M13 | 5′ CAG GAA ACA GCT ATG AC 3′ | M13 sequence |
a
Primer names are organized in groups. Prefixes: RT, reverse transcription primer; F, forward PCR primer; R, reverse PCR primer.
b
The T7 RNA polymerase promoter sequence in primer F20 is shown in italics. Restriction sites introduced by PCR are underlined and specified in parentheses at the end of the sequence. Silent mutations within the viral sequence are shown in boldface. Fragments a through f correspond to the letters in Fig. 1.