Table 3.
Oligonucleotides used for PCR amplifications and Q-RT-PCR analysis.
| Oligonucleotidea | Methodb | Oligonucleotide sequence 5′ → 3′c | Amplicon |
|---|---|---|---|
| XmaI-SP6p-G-5′TGEV-VS | PCR 5′TGEV | cgcgCCCGGGATTTAGGTGACACTATA(G)ACTTTTAAAGTAAAGTGAGTGTAGC | 557 bp |
| 5′TGEV-504-RS | PCR 5′TGEV | CACCAATGACGTAGTGATCCTTACG | |
| XmaI-T7p-GGG-ORF7-VS | PCR 3′TGEV | cgcgCCCGGGTAATACGACTCACTATA(G)GGATGCTGTATTTATTACAGTTTTAATC | 545 bp |
| HindIII-poly(A)-RS | PCR 3′TGEV | gccgAAGCTTTTTTTTTTTTTTTTTTTTTTTTTTGTATCACTATC | |
| TGEV-Leader-VS | Q-RT-PCR mRNA 7 | CGTGGCTATATCTCTTCTTTTACTTTAACTAG | |
| TGEV-7(38)-RS | Q-RT-PCR mRNA 7 | AAAACTGTAATAAATACAGCATGGAGGAA | |
| TGEV mRNA 7 (TaqMan MGB probe) | Q-RT-PCR mRNA 7 | FAM-CGAATCAAACGAGATGCT-MGB |
a
VS, virus sense. RS, reverse sense. MGB, minor groove binder group.
b
PCR 5′ (or 3′) TGEV, PCR for amplification of the TGEV genome ends to generate transcription templates. Q-RT-PCR mRNA 7, primers and probe used for the quantification of the viral subgenomic mRNA 7.
c
Restriction endonuclease sites used for cloning are in italics. Additional sequences at the 5′ of the restriction site are in lower case. Transcription promoters are in bold. Transcription initiation sites are in brackets. Template sequence corresponding to the poly(A) is underlined.