First I would like to thank all of those participating with their great posts and discussions in this wonderful forum.
I would like to check whether I'm getting the right results or not! As I'm not an expert in this field (It's really urgent).
My project is about checking the methylation status of candidate genes with a defined CpG island in Human cancer cell line and gDNA from matched tissue samples.
I found 5 candidates but no one have a previously reported MSP primers so I used Methyl Primer Express v1.0 (from ABI) to make the primers.
That is what I have done:-
1- I got promoter sequences from promoter database at: (I found promoter seq of only 1st four genes)
2- I BLASTn the promoter seq. to find whether it will match the candidate genes or not. (The result was OK!)
3- I insert the promoter seq. in Methyl Primer Express v1.0 and followed the software default (the default criteria is attached to the post).
4- I had the MSP primers for my genes (Attched)& for the last genes I also got the MSP primers (Attached) after using a method similar to that of pcrman (http://www.protocol-...small-rna-blog/)
Also I want to know if those primers are still OK if they don't have one of the following (found them in literature):-
A- 20-30 bp in length
B- GC content similar to that of the template.
C- Avoid streches of polypurines/polypyrimidines.
D- Distance between primers should not be more than 3kb.
E- Best to have mismaches at the 5' of the primers.
F- M primers must contain Cs in the CpG context at 3'.
G- U primers must have Ts located after Gs in 5' and 3'.
H- No complementarity at 3' of the F & R primers.
Finally, please accept my apology for such a long post.
Thanks in advance for your kind help,