Bisulfite-Treated Tyrosine Hydroxylase Gene Protocol

The following primer pairs were designed by BiSearch to amplify a fragment of the bisulfite-treated tyrosine hydroxylase (TH, D00269) gene from genomic template:

THbs114 GAGGTTTTGGTGTTTATTAAA;

THbas308 TAAAATCTAATTACCTTCACTCC;

THbs566 TGTTAAGTAGGTAGAGGTTATTAT; and

THbas827 AACCTAAAAAAAACACACAC.

The AmpliTaqGold amplification system (Applied Biosystems) was used for the PCR of bisulfite-treated DNA with standard cycling conditions after an initial cycle of 10 min at 95°C: 30 cycles with denaturation at 95°C for 30 s, annealing at 56°C for 30 s and elongation at 72° for 90 s. A second PCR program was also performed, which differed from the first one by the annealing temperature lowered to 49°C (as indicated in the Results section).