I am currently using the pCpGL-basic vector to evaluate the influence of methylation on a sequence that contains a single CpG site. I expanded my plasmid in E.Coli PIR1 as recommended, then I methylated the plasmid with SsiI and SAM, but when I checked the methylation status of both the methylated and unmethymated plasmids, I found out they are both methylated. I performed bisulfite sequencing to check them, and I was surprised also the Cs in non CpG sites are methylated! Discussing it with a bacteria expert I got to know that Bacteria tend to methylate plasmids.. so I am wondering whether we have always been working with methylation going around? Is anyone having the same problems? thanks for your replies.
Hello Benedetta,
The good thing to use pCpGL system is that all CpG sites are removed from the vector, so that it rules out the effect of methylation on vector itself. I don't think methy at non CpG sites will have affect on your function results. For me, after in vitro methylation, I simply check the methylation efficiency by digesting plasmid with PmiI, which only recognizes unmethylated CACGTG. You may find the suitable restriction enzyme sites for your sequence using Snake Charmer. http://insilico.ehu.es/restriction/two_seq/snake_charmer.html
Hi Benedetta,
you are right, most E. coli strains contain three site specific methylases. That means that in transient assays, we usually use methylated DNA. However there is no CG methylation. Therefore you may still study this specific site. If you want to reduce A-methylation at GATC, and C-methylation at CCAGG and CCTGG sites you may amplify your DNA in dcm-/Dam- strains.
The NEB web site provides a good summary:
https://www.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation
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