How to determine the DNA methylation of the whole gene - Primer design for BSP (Jan/25/2006 )
You should be aware that CAG and CTG methylation is common in plants. You might want to think about this in the design of your primers, and look for it in your results.
Thanks phage434, can you tell me how can I design primers for my plant gene sequence? Will I consider all the Cs as methylated and design primer with degenerate nucleotides (for C)?
If you are trying to amplify treated DNA, then you want to use primers which don't contain CG or CAG or CTG sequences. These will not select for modified strands, but will allow you to sequence modified bases located between these sites. Because only one strand is methylated, you will see a mixture of C and T at methylated C sites, and a mixture of G and A at sites where the C is methylated on the complementary strand. Essentially, you are amplifying a mixture of two different sequences -- the modified top strand and the modified bottom strand.
If you are trying to select for top strand sequences which have been converted, then the forward primer should convert CG sequences to TG, CAG sequences to TAG, CTG sequences to TTG, while the reverse primer should convert CG sequences to CA, CAG sequences to CAA, and CTG sequences to CTA.
The difficulty here is that you are guessing that these bases are really modified. The benefit is that you are selecting modified strands of DNA (in this case the top strand), which will be comletely modified C to T in positions where the there is complete methylation.
If you are trying to select for bottom strand sequences which have been converted, then the forward primer should convert CG sequences to CA, CAG sequencs to CAA, CTG sequences to CTA, while the reverse primer should convert CG sequences to TG, CAG sequences to TAG, and CTG sequences to TTG.
This is tricky and I'm not sure I've explained it well. I may also have gotten myself confused, but I think I've tried to be careful.
The Cs might be methylated or non methylated, at this moment I have no idea. Is it a good idea to design primers using degenerate nucleotides at the place of all the Cs?
I would like to check the sense strand for methylation analysis.
Thaks for your suggestion.
my boss would like to buy bisulfite DNA modification to analyze methylation in N. benthamiana, i was wondering if you could tell me which one works best for your samples?
I's really appreciate your help. I have tried to use DNA beads protocol posted on this forum but my DNA did not get converted.
Thanks a lot.
I used EZ DNA Methylation Kit from Zymo Research and it worked nicely. You can try this.
same for me. I have used the zymo kit for months and I have had a conversion rate of above 99% all the time.
thanks a lot. I will let you know how it works for me!