DNA methylation content (degree) - (Feb/10/2006 )
I am very confused with a concept. Could anybody tell me what methylation degree means in a promoter and how to calculate it ?
There is an sentence in one paper as followings: I can not understand it very well.
" The promoter was either unmethylated, fully methylated, or hemimethylated using 5-methl deoxycytidine CED phosphoamidite "
Would someone like to help me? Thank you so much!
basically it's can be calculated as the percentage of all CpG's within your promoter that are methylated.
Now depending on how you assayed for methylation by bisulfite PCR and sequencing, either directly sequencing the promoter or clone and sequencing the clones of your promoter, will depend on how you calculate the degree.
For clonging, So you count the total number of CpG's within your promoter sequence, then count the number of them that are methylated by bisulfite sequencing for each clone and then take the average, with CpG islands, they are usually invariably either hypermethylated (all methylated) or unmethylated (all not methylated). If they are hemimethylated, by cloning, this would suggest that one allele is hypermethylated while the other is unmethylated (or hypomethylated) and this will present as some clones being hypermethylated while others are unmethylated.
for direct sequencing, you take each CpG dinucleotide and measure the peak heights of C and T at the CpG sites in a chromatogram. To make things easier I have seen cut off ranges in 25% increments. so at each site it can be either 0%, 25%, 50%, 75% or 100% methylated.
There are also variations to these methods and they are not all set in stone.
Thank you , nick. That's a very detailed answer and does a great help to me!