User:Torsten Waldminghaus/Methylation analysis with qPCR
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- As independent method and first step one could analyse cutting by methylation sensitive REN with qPCR
- on whole chromosome
- clusters and isolated GATCs
- coding regions and intergenic regions
Possible restriction enzymes that are Dam methylation sensitive:
| Enzyme | Site | Notes | Link |
|---|---|---|---|
| HphI | GGTGA | No star activity; reaction at 37°C; heatinactivation at 20 min 65°C | http://www.fermentas.com/catalog/re/hphi.htm |
| MboII | GAAGA | Star activity; reaction at 37°C; heatinactivation at 20 min 65°C | http://www.fermentas.com/catalog/re/mboii.htm |
| TaqI | TCGA | No star activity; reaction at 65 °C; no heatinactivation after 20 min 80°C | http://www.fermentas.com/catalog/re/taqi.htm |
- HphI seems to be a good choice since it can be heatinactivated and has no star activity (does not cleave unspecific when DNA is overdigested).
- In unsynchronized cultures the detection of hemimethylation will be difficult. If every GATC is hemimethylated for about 1 min and replication of the chromosome takes about 50 min, than about 2% of a specific GATC will be in a hemimethylated state giving only 1% cut with an overlapping enzyme.
