MSP_Primer dimers? - (Apr/08/2015 )

Dear Nick,

 

thanks:) I don't know, I used 0, 0.1, 1, 10, 100% methylated standard. So, at least in the upper profile for example, the 100%, I should have only one peak I think, or? 

 

Elena 

..or what you mean is that my primers amplify another sequence and that the two peaks are proportional since I'm using standard between 0 and 100 % methylated and so, also the additional sequence I amplify has the same rate of methylation and, therefore, it shows the same kind of profile. 

But how is it possible that 300 pb melt before? Or is it possible that my peak is the first one and it could be that it is smaller because the sequence is shorter and so it has less CpG (in number)?

 

thanks a lot,

 

Elena

well how sure are you your standards are what they say they are? also, could it be that your 300bp template is unmethylated? if so, it would have a low GC-content and therefore low melt. 

 

N

I'm completely sure about my standards, because I converted them from the commercial fully methylated and fully unmethylated human DNA. The  300 pb also correspond to the same methylation rates because is also converted from these commercial standards, so it is not possible that the 300 pb is unmethylated. Furthermore, my MSP primer don't amplify unmethylated DNA fragments.. I really don't get what happen with my analysis.. also because the primers are theoretically specifics. What I could do is purify my 200 pb band from the gel and to HRM on it, to see at least to which peak correspond my gene... 

 

Elena

Hi Elena,

 

maybe if you feed your sequences into a melt simulator such as uMelt, then you can see, you can then feed in your bisulfite converted sequence and then your methylated and unmethylated sequences for good measure.

 

cheers

 

Nick

 

https://dna.utah.edu/

THANK YOU Nick. That is very interesting. I put my sequence in the uMELT and even the program give me 2 peaks. It's like if this sequence has two melting points. is it possible?

 

Elena 

hi again. Great, I just found the answer: 

https://eu.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2014/01/20/interpreting-melt-curves-an-indicator-not-a-diagnosis

 

Thank you very much again, Nick!!!!! I still have to optimize, but it has been really helpful discussing with you!!!!

 

Elena 

Great to see Elena, thanks for the IDT link, this would be useful to everyone else too. 

 

Cheers

 

N