methylnick.please help me with MSP primer designing! - (Dec/01/2008 )
I am new here ! I have to design MSP primer to solve the problem,The gene's sequence is as the attachment,the CpG sites for detection are underlined and highlighted with yellow,I have three question here:
1,For this particular sequence ,the best choice is MSP or BSP?
2,How can we design primer for msp ? method in detail,or is there any specification for it?
3,MSP'mechanism ? i have found many articles about it ,but without pictures , i can't know the detailed mechanism!
Thankyou for your answers !
1. For this one, you could do either one, MSP or BSP. BSP can give you detail information about each CpG methylation status, but cloning needed, and also more clones are needed to get accurate results although approximately 10 clones are usually sequenced. MSP is a cost-effective method, qMSP also can give you percentage of methylation.
2. Methylprimer software often is recommended, but I am not sure whether it works well or not. If need help, I may help design one.
3. You can gooogle to get more information about MSP. Basically, all unmethylated Cs will be converted to T with bisulfite treatment, but no change for the methylated Cs in CpG island. Importantly here, you have to treat your DNA with bisulfite first with kit (I recommend here, ZYMO research kit), and then run PCR.
thanks a lot for your reply. I have searched MSP so much , but it is so abstract to understand.Maybe a vivid procedure can solve the newbile'question. Are there any step-by-step instruments about MSP primer designing?
you can do this by MSP, but if you were to do it by BSP then you need to redesign your primers (so there are no CpG's in there). One or the other will depend on what you would like to find out. Yes or no for methylation MSP is the way to go, yes methylation but by how much? then you would go BSP.
ABI's Methylprimer Express works very well and is free, google that.
As for tools to learn MSP, there are ones around and i think methylprimer express has a good help section that explains this.