The perfect number of CpGs in MSP primer - (May/11/2005 )
I have had some problems with two pairs of MSP primers. I have designed all of them and contain five CpG dinucleotides each one more or less. I have tried to improve the MSP conditions but results in the electrophoresis gel are horrible. I have a lot of bands!
I have seen in some papers that primers contain two or three CpG dinucleotides. I designed mine with five because I thought it would be more specific, but now I have some doubts about it. What do you think?
I have decided to make another primers and I want to be sure that I have the correct number of CpG dinucleotides.
Thanks in advance.
That is a very good question and but there is no good answer to it.
If a MSP primer contains only one CpG 'C', the specificity may not be high compared to more than one CpG 'C', but it will definitely tell you the true methylation status of that single 'C'.
Usually neighouring CpGs are not methylated equally. If a MSP primer contains more than one CpG 'C's, we don't know how this primer will behave in this situation.
A third case, both forward and reverse primers contain a CpG 'C', if one 'C' is methylated and the other is not, what will you get from the pair of primers? Who knows.
To find a compromise between multiple CpGs and one CpG, I would say a primer should have 1-2 CpG 'C's. Anyway, the most important 'C' is the 3' most one.
Pcrman is right in saying the 3' C is the most important because a mismatch will prevent the polymerase from extending.
I have reservations in incorporating CpG's within the primer because you will never know what the methylation status of these will be in your system.
I would try and not include CpG's within the primer and ensure the primer has a high Tm, ideally close to the extension temperature of your polymerase to ensure extension is inhibited by the 3' mismatch. If this can not be avoided then I would select primers with CpG's but the C is a degenerate base Y (for C/T)
I guess valencia was talking about MSP primers which must have CpG 'C's in them, although both of us don't favor MSP.
oh okay my bad!
Thanks a lot for your answers.
I have designed new primers with two-three CpGs, and the Tm is 64ºC. I also have avoided to include so many "C" that will be converted in "T" in these primers.
I hope my MSPs will run better.
Thanks another time.
You still need quite a few non-CpG 'C's in your primers to tell apart modified from unmodified DNA.
Sorry for the delay.
Yes, of course I have non CpG "C" in the primers. A pair of them run really well, but the others show some extra bands on the gel.
I will try to improve the MSP reaction for them increasing the temperature of annealing and decreasing Mg concentration.
Thanks in advance.
good luck with it valencia, looks like you are on the right track.