Table 1 Primers for amplification and sequencing of UGT1A
| Primer name | Sequence (5' to 3') | Condition |
|---|---|---|
| Amplification of UGT1A1 transcriptional regulatory region for direct sequence | ||
| Amp1-F | CACCTCCTCCTTATTCTCTTa | fragment 1 |
| Amp1-R | CCTTGAATTTCCAAAATCCCAGA | 3 min at 96°C, 35 cycles (20 sec at 96°C, 15 sec at 57°C, 90 sec at 72°C), 5 min at 72°C |
| Amp2-F | GATACAAGGCAGAACAGAAC | fragment 2 |
| Amp2-R | AGGTCACACGGTTACTCTGA | 2 min at 94°C, 35 cycles (20 sec at 94°C, 30 sec at 61°C, 70 sec at 72°C), 5 min at 72°C |
| Amp3-F | TGAGCGCTGAAAATCTCAAC | fragment 3 |
| Amp3-R | AGAGAGGAAGAAGGACGACT | 3 min at 94°C, 32 cycles (30 sec at 94°C, 45 sec at 59°C, 60 sec at 72°C), 10 min at 72°C |
| Amp4-F | ACAGGTTTCCATGGCGAAAG | fragment 4 |
| Amp4-R | GCTTGCTCAGCATATATCTCTGGGb | 2 min at 94°C, 35 cycles (20 sec at 94°C, 30 sec at 61°C, 60 sec at 72°C), 5 min at 72°C |
| Amplification of proximal and distal regulatory regions from genome DNA for cloning | ||
| Proximal-F1 | GACTGCCATCCAGTAGGGCTCACACGTT | |
| Proximal-R1 | CGCCTTTGCTCCTGCCAGAGGTTCG | 2 min at 94°C, 30 cycles (20 sec at 94°C, 5 min at 68°C), 10 min at 68°C |
| Distal-F1 | GAGATCTGAGTTCTCTTCACCTCCTCCT | |
| Distal-R1 | GCAGAGCTTCCAAGCTTTTTGAGGCTG | 2 min at 94°C, 30 cycles (20 sec at 94°C, 30 sec at 63°C, 2 min at 72°C), 5 min at 72°C |
|
Addition of restriction enzyme site of cloned transcriptional regulatory regions by PCR (underline: Sac I site on Proximal-F2 and Distal-F2; Xho I site on Proximal-R2) | ||
| Proximal-F2 | GAGAGCTCCCTCAGCCCCTAGAGCACCATC | |
| Proximal-R2 | CTCTCGAGGCGCCTTTGCTCCTGCCAGAGG | 2 min at 94°C, 8 cycles (20 sec at 94°C, 30 sec at 60°C, 90 sec at 72°C), 5 min at 72°C |
| Distal-F2 | GAGAGCTCGAAGGGATTAGTTTAGGACAACCCTCCTTC | |
| Distal-R1 | GCAGAGCTTCCAAGCTTTTTGAGGCTG | 2 min at 94°C, 8 cycles (20 sec at 94°C, 30 sec at 60°C, 90 sec at 72°C), 5 min at 72°C |
| Sequencing primers for transcriptional regulatory region of UGT1A1 | ||
| Seq1-1 | TATTCTCTTTTTGACACTGG | |
| Seq1-2 | GACCAAGGTTCCAGAAGTGGTGGTGA | |
| Seq1-3 | CAATTACAGGGGATGGTGCTCTAG | |
| Seq1-4 | CTTCCAATTCTGGCTGCACA | 5 min at 96°C, 25 cycles (10 sec at 96°C, 5 sec at 50°C, 4 min at 60°C) c |
| Seq1-5 | GACGAAGGAATGAAACACAT | |
| Amp4-F | ACAGGTTTCCATGGCGAAAG | |
| Sequencing primers for cloned transcriptional regulatory regions | ||
| Seq2-1 | TCTGCTGTTGGCTGAATCTG | |
| Seq2-2 | TATACACACGGCCTGCAAGT | |
| Seq2-3 | CAGAATGGCTAGAGGGTAAG | |
| Seq2-4 | ACAGAAACATGTCCAGAGCACTT | |
| Seq2-5 | TGTCTGATGGTGGCCTACTA | |
| Seq2-6 | TTGCTGCCCTGCTGTGTG | 5 min at 96°C, 25 cycles (10 sec at 96°C, 5 sec at 50°C, 4 min at 60°C) c |
| Seq2-7 | CATCCAGGTACACAGCAGAA | |
| Seq2-8 | TCATTCCACTGGCCCAAGAT | |
| Seq2-9 | GGTTCCCAATCAGGTCCATT | |
| Seq2-10 | TCACATGCGCTCCAGTGAAT | |
| Seq1-2, Seq1-4, Amp4-F | ||
