2 replies to this topic

[mulain]

Hello everyone!

I want to carry out MSP for a gene that is located on the reverse strand. And thats what finally got me thinking:

• Methylation occurs at CpGs, so does it occur for each strand separately? A CpG in the forward strand would be a GpC in the reverse, meaning the C would not be methylatable. On the other hand, forward-strand GpCs would be CpGs in the reverse strand, making them potential methylation targets.
  
  
• If the strands methylation differs, which strand's methylation is relevant (or both)? In the past I have always looked at forward strand methylation, but now I have a gene on the reverse strand. How should I design my primers? To include forward-strand CpGs or reverse-strand CpGs?
  
  
• Is there any literature on this? I haven't found anything! This must have been investigated at some point!

I'm pretty sure I'm just missing something but I haven't been able to find it by myself... so someone please enlighten me  

cheers
mulain

[pcrman]

• Methylation occurs at CpGs, so does it occur for each strand separately? A CpG in the forward strand would be a GpC in the reverse, meaning the C would not be methylatable. On the other hand, forward-strand GpCs would be CpGs in the reverse strand, making them potential methylation targets.
  
  Most methylation is symmetric and occurs to both strands, but only to the cytosine that is before a G (though recently it was found the non-cpg c methylation is not uncommon).
  
  for example:  
  
                 forward      .....C^mG.......
                 reverse      .....G C^m.......
  
  * C^m:methylated cytosine.
  
  
• If the strands methylation differs, which strand's methylation is relevant (or both)? In the past I have always looked at forward strand methylation, but now I have a gene on the reverse strand. How should I design my primers? To include forward-strand CpGs or reverse-strand CpGs?
  
  You could design primers using either strand as template. The result will be the same. The PCR product will be the same too.
  
  
• Is there any literature on this? I haven't found anything! This must have been investigated at some point!

There is, but I could not remember off the top of my head.

[mulain]

Thanks a lot, pcrman! Now I can go ahead and design my primers
A great help, as always!