FMO3 template amplification primers for SNP discovery
Coordinates are based on assigning the “A” of the ATG translation initiation codon +1. All PCR conditions. (A, B, C, D) had an initial denaturation step at 94°C for 2 min followed by 35 cycles of: A, denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 1 min; B, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 1 min; C, denaturation at 94°C for 30 s, annealing at 47°C for 30 s, and elongation at 72°C for 1 min; D, denaturation at 94°C for 30 s, annealing at 48°C for 30 s, and elongation at 72°C for 1 min. All PCR conditions (A-D) had a final elongation step at 72°C for 2 min.
| Primer Set | Primer Sequence | FMO3 Coordinates | PCR | Target |
|---|---|---|---|---|
| 1 | 5′-TTCCTGGTACTAATAGATCA-3′ | −2310 to −2291 | A | Upstream |
| 5′-TCTGTGTGTCTACGTCCT-3′ | −1716 to −1733 | |||
| 2 | 5′-ATCCTCTAATCCTTGTTAAA-3′ | −2733 to −2714 | A | Upstream |
| 5′-CCAATAAGGAGGATGACT-3′ | −2145 to −2162 | |||
| 3 | 5′-TGGGAGACTGGCCTACAG-3′ | −1906 to −1889 | B | Exon 1 |
| 5′-GAGACGGAGTTTCGCTTTTA-3′ | −1455 to −1436 | |||
| 4 | 5′-ATTAAGCCAAAGAGCGAAAT-3′ | −102 to −83 | B | Exon 2 |
| 5′-TTTCAAAGCCCATTGTCTAA-3′ | +334 to +315 | |||
| 5 | 5′-GCCCTGACCATGATCAGTAT-3′ | +11062 to +11081 | B | Exon 3 |
| 5′-GAGAGGCCACACTGTTAAA-3′ | +11442 to +11424 | |||
| 6 | 5′-ATATTTTTCTTAACCCAC-3′ | +14920 to +14937 | C | Exon 4 |
| 5′-TAATTTAACCTGCTTATACT-3′ | +15255 to +15274 | |||
| 7 | 5′-CATCTATTCACAAGGTCGC-3′ | +15336 to +15354 | B | Exon 5 |
| 5′-CCCCACATTTCATATCACAC-3′ | +15646 to +15627 | |||
| 8 | 5′-CTGCAGCTGGGGTAATAG-3′ | +18032 to +18049 | B | Exon 6 |
| 5′-CCAGCAGGCATATCACGTTT-3′ | +18371 to +18352 | |||
| 9 | 5′-ATTTATATATGGACCAAT-3′ | +21276 to +21293 | D | Exon 7 |
| 5′-GACCTTGTAACTAGGATTAT-3′ | +21796 to +21777 | |||
| 10 | 5′-TAGAACTGAATTTGGTGTCT-3′ | +23412 to +23431 | A | Exon 8 |
| 5′-GCTTGTAGTTGTCATTCC-3′ | +23743 to +23726 | |||
| 11 | 5′-TGTTGTATGTCAGGGTAGT-3′ | +24288 to +24306 | B | Exon 9 |
| 5′-CTTTCAGAAATCCTAGAGAA-3′ | +24810 to +24791 |