Thermopol taq polymerase

Methylated DNA was analyzed using the differential methylation hybridization technique and was co-hybridized to Agilent Human CpG Island Microarrays at the University Health Network Microarray Centre according to the manufacturer's recommended protocol. Genomic DNA (100-200 ng) from bladder tumors was first digested with MseI. H-12/H-24 linker oligonucleotides were annealed together, creating overhangs that bind MseI-digested DNA. DNA was then ligated to the MseI cleaved ends using T4 DNA ligase and overnight ligation at 4°C. Ligated DNA was then sequentially digested first with HpaII followed by BstUI, both of which are methylation-sensitive enzymes. Amplification of intact fragments was then performed with ThermoPol Taq polymerase (New England Biolabs, Pickering, ON, Canada) with the following conditions: 72°C for 5 minutes, 95°C for 1 minute, 24 cycles of 95°C for 1 minute followed by 67°C for 2.5 minutes, and a final step of 72°C for 5 minutes. Following PCR, DNA was purified using the QIAquick PCR purification kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer's instructions. Reference DNA, which was co-hybridized with tumor DNA to microarrays, was prepared in a similar fashion using lymphocyte DNA pooled from six healthy age-matched men.