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anjama 03-10-2017 11:02 AM

PstI methylation sensitivity
 
So my lab is in the process of understanding and implementing a protocol for several species. One point of confusion we are trying to figure out is whether or not the PstI restriction enzyme is methylation sensitive. The reason this is a concern is that the protocol calls for methylation sensitive enzymes, and people involved in developing the protocol use PstI as a methylation-sensitive enzyme for a variety of species ranging from plants to mammals. However, the product pages for Fisher and NEB both list PstI as not sensitive for dam, dcm, and CpG sites. The only indication I can see on manufacturer sites that it is sensitive is a reference chart on Promega's website that lists it as sensitive to CpNpG methylation sites.

Just taking a quick look through primary literature, many papers seem to refer to PstI as methylation sensitive, but I've also seen some that refer to it as non-sensitive. From what I gather, CpNpG sites are predominately a plant thing, whereas the mammal genome consists of primarily CpG sites (I think I saw the number 97% somewhere), which seems like it explains the discrepancy between different papers. Is this right? Is it correct to consider PstI non-methylation sensitive for mammals, but methylation sensitive for plants? Or am I missing something? Thanks

SNPsaurus 03-10-2017 05:15 PM

For RAD-Seq work in plants, PstI acts as if it is methylation-sensitive, as you'll get a number of cut sites sequenced that is consistent with the genome being several hundred megabases in size, regardless of the actual genome size (plants have a small amount of non-repetitive DNA with most of the variation in size coming from methylated repeats).

PstI cut sites tend to scale with genome size in mammals, so it acts insensitive there. These are not direct tests of sensitivity, of course, but it is consistent with the enzyme companies sites. So you are correct, I'd say!


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