PCR more difficult in methylated CpG region? - (Nov/17/2004 )
I am trying to check the methylation status for a gene promoter containing CpG island in cancer cells. my three sets of primers works perfectly fine with the untreated genomic DNA from cancer cell lines and testis tissue(supposed to be hypo-methylated), but I never made them work on somatic tissues such as liver and placenta (supposed to be hyper-methylated). genomic DNA quality looks good on the agarose gel(high molecular weight, not obvious degradation). Does anyone have encountered the same situation? any clue to solve it?
Any information is appreciated!
Do you mean amplifying unmodified DNA or bisulfite modified DNA?
I often found that methylation PCR works better with DNA from cell lines than DNA from tissue for unknown reason.
I mean the untreated genomic DNA from somatic tissues. If the hypermethylation in the CpG region do interefere the efficay of PCR, bisulphite treatment may help because the possible secondary structure may be detroyed by bisulphite. I am doing PCR on treated DNA, hopefully, I will get my PCR work on treated DNA even it did not work on untreated condition.
We know that GC rich regions are hard to amplify. If your observation that methylated regions are refractory to PCR is true, that would be an interesting finding.
I also have the problem that DNA from cell lines works better than DNA from tumor tissues despite DNA quality looks good!
Did you ever find a way to improve the results on DNA from tissue?
Thanks in advance!
Use JumpStart Taq from Sigma.
I am also studying the methylation of a gene promoter. For the past couple of months I've been struggling to amplify this region by PCR. We've designed multiple pairs of overlapping primers. I thought that since all of the components in the PCR were good that it must be a problem with the original sequence. Now its somewhat comforting to hear that I'm not alone. I've since diverted myattention to protein expression because of the difficulty. Still haven't found a good way to amplify the region.
Have you tried lowering the *extension* temperature? Try a 64C extension rather than 68 or 72, and lengthen the extension time a bit.