methylation specific PCR - Primers - (Sep/16/2010 )
I am currently interested in comparing DNA from a study organism(fish), before and after treatment with demethylating agents and hdac inhibitors and am in the process of designing my experiment. I would like to convert the DNA using bisulfite conversion and then I would like to perform methylation specific PCR on the products to identify areas of differential methylation and then I want to run the pcr products on a gel and sequence the resulting bands. This has become complicated by the fact that I am relatively new to methylation and genetic studies. My question is this: When performing msPCR the target genes are generally known and primers can be designed for the target genes easily, but I want to amplify the entire methylome/epigenome during PCR (because I do not yet know any target genes for the study organism)in order to run the methylome/epigenome on a gel for later sequencing. What primers should I use? How would I design them? Do they have to be targeted for specific genes or can non-specific primers be created? How do i amplify the entire genome for my purposes?
Any help is greatly appreciated.
Hey, I did not quite understand what exactly you wanted, but found something by chance today.
here, http://www.appliedbiosystems.com/absite/us/en/home/support/software-community/free-ab-software.html, you will get a software to design methylation specific primers....
hope it helps....