Background
Methods
Sequencing of the UGT1A1 transcriptional regulatory region
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 |
Construction of expression vectors
Cloning of the regulatory region in two separate DNA fragments
Construction of expression vectors
type | Number of allele | gtPBREM | Polymorphisms in the region between gtPBREM and TATA box | TATA box | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
JRSa
(100) e
| JG7b
(8) e
| NCc
(20) e
| CGd
(22)e
| -3275 | -3152 | -2951 | -2743 | -2737 | 2726 | -2724AT[n] | -2473 | -1352 | -1125 | -997 | -689 | -364 | A(TA)nTAA | |
Haplotypes with A(TA)6TAA | ||||||||||||||||||
I | 68 | 0 | 11 | 0 | T | G | A | T | T | G | 3 | T | A | C | G | A | C | 6 |
II | 19 | 0 | 4 | 0 |
G
| G | A | T | T |
A
|
>8
|
G
|
C
| C |
A
|
C
| C | 6 |
III | 0 | 0 | 1 | 0 | T | G | A | T | T | G | 3 |
G
|
C
| C |
A
|
C
| C | 6 |
IV | 2 | 0 | 0 | 0 | T | G | A | T | T | G | 3 | T |
C
| C |
A
| A | C | 6 |
Haplotypes with A(TA)7TAA | ||||||||||||||||||
V | 10 | 8 | 0 | 0 |
G
|
A*
|
G*
|
C*
|
C*
|
A*
|
8*
|
G*
|
C*
| C | G |
C*
|
T*
|
7
|
VI | 1 | 0 | 3 | 12 |
G
|
A
|
G
|
C
|
C
|
A
|
>8
|
G
|
C
| C | G |
C
|
T
|
7
|
VII | 0 | 0 | 1 | 5 |
G
| G |
G
|
C
| T |
A
|
>8
|
G
|
C
| C | G |
C
|
T
|
7
|
VIII | 0 | 0 | 0 | 4 |
G
| G |
G
|
C
|
C
|
A
|
>8
|
G
|
C
|
T
| G |
C
|
T
|
7
|
IX | 0 | 0 | 0 | 1 |
G
|
A
|
G
|
C
|
C
|
A
|
>8
|
G
|
C
|
T
| G |
C
|
T
|
7
|