Are my MSP primers good? - MSP primers design by Methyl Primer Express v1.0 (Jun/23/2011 )
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-online.org/forums/blog/4/entry-10-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,
I think if you give the right input sequences to the program, you can rely on it to pick the primers for you and then test them. So for your part, you just need to get the right promoter sequences and let the program takes care of the design.
For the fist 2 genes, the sequence is on the first exon (though it is on a cpg island).
For NKx2-3 gene, the sequence is too far away from the promoter
For CLEC3B, the sequence is a mRNA sequence of the gene, not DNA sequence at all.
Thank you very much for the kind reply,
But what about the 4th gene?
I'll try to fix the NKX & the last gene but this will take some time as am a little busy these days.