TABLE 2.
Primers used for reverse transcription and PCR amplification of HCV RNAa
| Region | Primer setb | Polarity | Sequencec | Annealing temp (°C) |
|---|---|---|---|---|
| IRES | Outer | + | 5′-GGGGCGACACTCCACCAT-3′ | |
| − | 5′-GCACACCCAATCTAGGGCCCCTGCGCGG-3′ (RT) | 53 | ||
| Inner | + | 5′-CACTCCACCATGGATCACT-3′ | ||
| − | 5′-GGAACTTGACGTCCTGTGGGC-3′ | 53 | ||
| 1aEcoRI (1) | + | 5′-cggaattcGCCAGCCCCCTGATGGGGGCGACACTCCACCAT-3 | ||
| 3aEcoRI (3) | + | 5′-cggaattcGCACCTGCCTCTTACGAGGCGACACTCCACCAT-3′ | ||
| UEcoRI | − | 5′-ctggaattcCGGGAACTTGACGTCCTGTGGGC-3′ | 50 | |
| E2/HVR1 | Outer (1) | + | 5′-GGGATATGATGATGAACTGG-3′ | |
| − | 5′-TCCCACCACCACGGGGC-3′ (RT) | 52 | ||
| Outer (3) | + | 5′-GCTTGGGATATGATGATGAACTGGTC-3′ | ||
| − | 5′-GGTGTGGAGGGAGTCATTGCAGTT-3′ (RT) | 60 | ||
| Inner | + | 5′-CACTGGGGAGTCCTGGCG-3′ | ||
| − | 5′-CGAGTGCTGTTGATGTGCCA-3′ | 58 |
a
PCR were carried out at the annealing temperature shown over 35 cycles.
b
Genotype-specific primers are indicated by the genotype in parentheses. RT, primers used for reverse transcription. 1aEcoRI, 3aEcoRI, and UEcoRI are primers which were used to amplify IRES sequences of interest for the purpose of cloning into the dicistronic vector.
c
The EcoRI restriction site and non-HCV nucleotides are indicated by lowercase.