Oligonucleotides used for the production of promoter fragments, mobility shift assays, site-directed mutagenesis, quantitative PCR, and bisulfite PCR Regarding the oligonucleotides used for the mobility shift assays and site-directed mutagenesis, the HNF1-motif in the hURAT1 promoter region is underlined. Boldface type indicates the difference in the sequence of the per and mut compared with the wild-type sequence found in the hURAT1 promoter.
Oligonucleotide | Orientation | Sequence (5′ to 3′) | ||
|---|---|---|---|---|
| Primers used for the cloning of 5′-flanking regions | ||||
| hURAT1 | ||||
| h – 253 | Forward | CGGGGTACCTTGGCTCAGCCACTCTGGGAGGT | ||
| h + 83 | Reverse | CCCAAGCTTAGAGAGGCAGCTGCTCCAGACC | ||
| mUrat1 | ||||
| m – 261 | Forward | CGGGGTACCCTGGGTTAGCTCACAGTACAG | ||
| m + 80 | Reverse | CCCAAGCTTAGCGACACAGCAGAGTCTG | ||
| Oligonucleotides used for the construction of EMSA probe and competitor or site-directed mutagenesis | ||||
| wt | Sense | CTCAACGCGGGTTAAACTTTGACCAAGGAAATG | ||
| per | Sense | CTCAACGCGGGTTAATCATTAACCAAGGAAATG | ||
| mut | Sense | CTCAACGCGGGGCGAACTGTGACCAAGGAAATG | ||
| Primers used for quantitative PCR | ||||
| mUrat1 | Forward | GAGGGAGACACGTTGACCAT | ||
| Reverse | AAGTCCACAATCCCGATGAG | |||
| mGapdh | Forward | AACGACCCCTTCATTGAC | ||
| Reverse | TCCACGACATACTCAGCAC | |||
| Primers used for bisulfite PCR | ||||
| mUrat1 | ||||
| –419-B-F | Forward | GGGAATTAATAAGGGAGTGTAGGAA | ||
| + 134-B-R | Reverse | TCACCATAAAACCTAAAACCCTCT | ||
EMSA, electrophoretic mobility shift assay