I want to study the methylation function on the promoter activity. I first methylate the promoter DNA by MSSS1(NEB). Then I transfected the methylated DNA into cells for reporter gene analysis.
My question is
1. How can I confirm whether my DNA methylation is full? The restriction endounuclearase e.g Hpa2 site is CCGG instead of CGCG.
2. I have constructed the reporter gene plasmid driven by my promoter. Whether I methylate the whole plasmid or only the promoter region?
3. and after I have methylated the DNA, whether I transform the methylated DNA directly transfect the cells? Can I firstly transform the methylated DNA into Ecoli to amplify the plasmid? Does the amplification in Ecoli can affect the DNA methylation status?
0
2 replies to this topic
#2
pcrman
-
- Global Moderators
-
- 1,025 posts
Epigenetist
46
Excellent
Excellent
Posted 06 February 2009 - 07:24 PM
This assay is very tricky and risky. There is no guarantee that the methylation will remain after you transfect the plasmid into the cells.
#3
methylnick
-
- Global Moderators
-
- 245 posts
Veteran
3
Neutral
Neutral
Posted 10 February 2009 - 01:07 AM
pcrman, on Feb 6 2009, 08:24 PM, said:
This assay is very tricky and risky. There is no guarantee that the methylation will remain after you transfect the plasmid into the cells.
It is almost impossible to do this enzymatically, there are a couple of tricks that you can do using site specific methylases like HpaII and HhaI methylases which methylate different recognition sites. There is no way to be sure what construct was transfected into cells (ie: how much methylation as pcrman alluded to)
If the promoter is not so big, you could synthesise this from scratch but is a very expensive way to go about it.
Nick
