Table 1 Primers for amplification and sequencing of UGT1A

Amplification of UGT1A1 transcriptional regulatory region for direct sequence

   Amp1-F

CACCTCCTCCTTATTCTCTT^a

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

GCTTGCTCAGCATATATCTCTGGG^b

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