Table 1.
Primers used in the bisulfite-converted cccDNA amplification
|
BSP primers:
| |||
|---|---|---|---|
| Target | Primers, 5′-3′ | Position (bp) | |
| CpG1 | 53–283 | ||
| Nested PCR options: ① f2 + down f2 + r2 ② f2 + down 1′ f2 + r2 ③ ViveF + down1′ viveF + viveR | |||
| CpG1-BSP-f2 | TATTTTTTTGTTGGTGGTTTTAGTTT | 54–77 | |
| CpG1-BSP-r2 | TAAAAAATTAAAAAAAATCCACCA | 286–266 | |
| CpG1-BSP-seminested downstream | ACAAAAAAATAAAACATAACAACAAAATA | 439–411 | |
| CpG1-BSP-semidown 1′ | AACAACATACCTTAATAATCCAA | 470–448 | |
| Primers from reference [16]: | |||
| Vivekanandan-CpG1-F | TTTGTTGGTGGTTTTAGTTTAGGA | 58–81 | |
| Vivekanandan-CpG1- R | TCCCCCTAAAAAATTAAAAAAAATC | 288–264 | |
|
| |||
| CpG2 | 1195–1666/1389–1666 | ||
| Nested PCR options: ① f2 + down f2 + r2 ② f2 + down CF2 + CR2 ③ CF2 + CR2 f3 + r2 | |||
| CpG2-BSP-f2 | GTAATTTTTATTGGWTGGGGTTTGGT | 1048–1069 | |
| CpG2-BSP-r2 | ATCCTCTTATAYAAAACCTTAAACAA | 1680–1655 | |
| CpG2-BSP-seminested downstream | TTATACCTACAACCTCCTAATACAAAA | 1794–1768 | |
| CpG2-CF2 | TTTTATGGTTGTTAGGDTGTGTTGTT | 1052–1077 | |
| CpG2-CR2 | TAACCTAAHCTCCTCCCCCAAC | 1762–1741 | |
| CpG2-BSP-f3 | TGTGTTGTTAATTGGATTTTG | 1389–1409 | |
| Primers from reference [13, 16, 17]: | |||
| Vivekanandan-CpG2- F | GTAATTTTTATTGGTTGGGGTTTG | 1194–1217 | |
| Vivekanandan-CpG2- R | TCCAATTAACAACACAHCCTAACAAC | 1404–1379 | |
| Kim-CpG2-F | GGGATTGATAATTTTGTTGTTTTTTT | 1329–1354 | |
| Kim-CpG2-R | TCCAAAAATCCTCTTATATAAAACCTTAA | 1672–1644 | |
| Guo-CpG2-F | GTTTTTTGTTTATGTTTTGTTGG | 1418–1440 | |
| Guo-CpG2-R | AAATAAAACAAAATACACACAATCCG | 1598–1573 | |
|
| |||
| CpG3 | 2206–2461 | ||
| Nested PCR: up + r2 f2 + r2 | |||
| CpG3-BSP-f2 | GTGGTTTTATATTTTTTGTTTTAT | 2208–2228 | |
| CpG3-BSP-r2 | AAAATACTAACATTAAAATTCCCAAA | 2460–2435 | |
| CpG3-BSP-seminested upstream | GTTATGTTAATGTTAATATGGGT | 2162–2184 | |
|
| |||
| MSP primers (primers from reference [15]): | |||
| CpG1-MSP-F | ACGTGTTTTGGTTAAAATTCGTAGTTTTTA | 292–322 | 95 °C 5 min (95 °C 10s, 55 °C 30s, 72 °C 10s) × 50 cycles |
| CpG1-MSP-R | AATATAATAAAACGCCGCAAACACATC | 376–402 | |
| Probe: [6FAM] GTTTTTTAATTTGTTTTGGTTATCGTTGGATG [BHQ1] | 348–379 | ||
| CpG2-MSP-F | TGTCGTTTCGGTCGATTAC | 1502–1520 | 95 °C 5 min (95 °C 10s, 52 °C 30s, 72 °C 10s) × 45 cycles, Melting curve, 40 °C 30s |
| CpG2-MSP-R | CACGATCCGACAAATAAAAA | 1560–1579 | |
| SYBR Green | |||
| CpG3-MSP-F | GTGTGGATTCGTATTTTTTTC | 2270–2290 | 95 °C 10 min (95 °C 10s, 53 °C 30s, 72 °C 10s) × 45 cycles |
| CpG3-MSP-R | GACGATTAAAACCTTCGTCT | 2393–2412 | |
| Probe: [6FAM]AACCTACCTCGTCGTCTAACAACAAT[BHQ1] | 2339–2364 | ||