Sequencing of MSP products? - (Jun/30/2008 )
I have only used BSP to analyze methylation. Now I want to look at a gene that has so many CpG sites that I can't design BSP primers... I have looked at publications and people seem to use MSP and then sequence the MSP products for this gene. Is this really a good strategy? MSP can of course be used but to sequence the products - isn't that a bias in that strategy? Or does it make sense to you?
Thanks in advance
you are absolutely right molbio you can't sequence MSP amplicons because of the biases due to primer design, can you point to some examples in the literature? This would be a big boo boo.
Maybe I don't understand it correctly, but to me it seems like they have sequenced MSP products in this article;
Kim et al, Cancer Res. 2005 Oct 15;65(20):9347-54
there are other examples regarding this gene...
this can't be right but not surprising that it has been published.
The correct way to perform sequencing is to use primers that are not biased to the methylated or unmethylated template, to directly sequence an MSP amplicon is only asking for trouble from the reviewers!!!
that was what I thought...
So, the best way to analyze a gene for methylation where you can't design primers for BSP is to do normal MSP visualized on gel - right?!
yes, but most cases you should be able to design MSP primers for your region of interest,