ABI have released MethylPrimer Express! - A new primer design program for DNA methylation....For Free!!! (Jun/02/2006 )

I'm not in this field, so my question may be a little basic... I'm using MPE to design MSP primers. Should I import the promoter sequence only or include the whole first exon region as well, since CpG are prevalent in both the promoter and first exon regions ?? they came up with some different results.
Thank you very much for the help~~

Glo

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glo,

I would say both promoter and exon as CpG islands at the 5' end of genes usually spans both, however the methylation of which CpG's is dependent on the gene and not the same for all genes so it would be best to test the whole region to see what the case is for your gene of interest.

good luck

Nick

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Thank you for the answer~ and also I came across a paper using real-time MSP with SYBR green, and then they cycle sequenced the PCR products. which one would be better comparing with BSP ?? My samples are human cancerous tissues, is it really necessary to do nested PCR ??

Glo

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which paper is this Glo?

are you sure they sequenced the MSP products? I think that would be a fatal flaw in the study as the primers are biased to either methylated or unmethylated DNA depending on the primer set used.

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Hi everybody,

we have tried this program to design primers for two regions which were difficult to get good BS-PCR reactions from: 100% succes.

greetings

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Hi @ all,

I´m quite new in this field and i have some quetsions. Hope that one of you can help me.

I try to find some good bisulfite primers for a repetetive sequence (IAP´s) and i´d like to do a nested PCR as suposed in other posts before.
My Problem here is that there are many CpG´s also in the surounding sequence so that i (and the MethylPrimer Sofware ) could not find 2 perfect primerpairs.

So what would you suggest, would it makes sense to do a hemi-nested PCR or try direct a "normal" PCR with only one Primerpair?

Another thing: I like the program so far, but how can i search for nested primers? Is there any feature i have not found yet, or do i have to try around with the lenght of the fragmet ( this is in the options for Primer design)?

Thanks for helping me !

dani

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Hi, Dear Nick:

I have downloaded the software and searched MSP as well BSP primers. It turns out that only the unmethylated primers for three genes worked, but all the methylated primers didn't. could you please give some advice?

Thank you very much in advance!

Best regards

dongdong

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Hi dongdong,

I guess you are talking about MSP - did you use methylated templates to establish the primers?

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I predicted the CpG island of my gene from 'webgene' as well as UCSC. They are over lapped. Thus I think the CpG island should be right. And then, I pasted this CpG sequence (without any change) into the window of Methylprimer Express and did as its wizard' guide. I selected some primers provided by this software. It turn's out to be the result I said in the last post. I curiously find that my interested genes are unmethylated both in the normal and cancer samples, while my RT-PCR test showed that only normal tissue expressed this gene but cancer tissue not. these results are not consistent. Could u please tell me what's wrong with it?

dongdong

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Hi dongdong,

sorry, I meant the DNA template. To optimize the PCR for the methylated primers it is adivsable to use fully methylated DNA that was bisulfite modified as template.
If your primers (U&M) work efficiently, there are other explanations for your result (no change in detected methylation but different expression). I would mainly guess that it's a methodological problem, as MSP detects only the methylation within the primer binding sites! So, MSP findings do not necessarily reflect the methylation status of the rest of the CpG island. MSP can be used as a screening tool for high-throughput analysis, but the relevance of the CpG's analyzed by your assay has to be confirmed by BSP or pyroseauencing first.
Hope that helps, Krümel

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