MSP and BSP confusion - a bit confusion about MSp (Jun/29/2007 )

Hi everyone
i am new in this area but befor posting this question i came across many valuable informations, but still i have a bit confusion in regard with the MSP-PCR. how many CpG site one primer can have at maximum? should the number of CpG sites in forward and reverse primer be the same? what about the CpG site in between the two primers? can they be be determined by sequencing, if yes direct or will need cloning first? and if one can find by this way than what is the difference between MSP and BSP?
hope no one will mind as i am very new in this field and have no experience but have to start working in a project. regards and thanx in advance.

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I'd say up to three CpGs. But given the nature of MSP it just doesn't make much sense to include more than one, as you are loosing all DNA sequences that are not equally methylated (you only see 3 methylated vs. 3 unmethylated but not 2 methylated or 2 unmethylated).
In my experience this is not mandatory. And can be even more senseful to use a non-specifc reverse primer without CpG.
You can, but the results are heavily biased by your primers which are specific for methylated or unmethylated DNA. I would regard sequecing of MSP products as serous flaw in a the study design. If you want to analyze all CpGs in your sequence you have to do BSP or pyrosequencing.
MSP is a rough method to estimate the methylation of 1-5 (at max) CpGs in the primer binding sites. BSP is the possibility to quantify all CpGs in a given sequence and is much more accurate.

Hope that helps, K.

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thanx krümelmonster' for ice reply
i have two more questions
1- what is the best way to sequence BIS-PCR product, direct or after cloning? what are the chances if i am going to do direct sequencing?
2-If there are conserved sequences in mouse in human, with known functions in mouse, should or must these sequences have the same functions in human?
Thanks once again in Advance

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1. cloning is muc more work but you have less trouble with the sequencing + you can automate the analysis. Direct cycle sequencing is less work, but the analysis of trace data takes some time and you will probably fail to get good readings in every case. Still, I prefer the latter, as I don't believe that cloning and sequencing really provides quantitative data (as long as you are not sequencing >50 clones). And, direct cycle sequencing is cheaper.

2. No, sequences can be conserved but have a different function in a different context. But think, in most cases in close relatives (like mammals) the function is likely to be the same. But I am no expert in this field.

K.

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Thank you Krumelmonster once again.
w007

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