In search of universal and reliable controls for methylation analyses - (Jan/27/2012 )
I´m trying to analyze promoter regions of an mammalian organism without published methylome data by bisulfite
pcr and methylated dna enrichment. Both methods require knowledge of at least one highly methylated
and one non-methylated genomic region to use as positive and negative control, respectively. As for the
negative control, in all organisms beta actin promoter (if containing a cgi, please correct me, if wrong) should be
reliable enough, i suppose. But: Do genes exist whose promoters are methylated in most mammalian organisms (independent of
cell type) in all likelihood? The ones i know are H19 ICR and IAP (commonly used for mouse and human), but
since the genomic sequence of my organism isn`t completely annotated, i need to find other regions for my purpose.
I hope you can make a proposal, because i`m really desperate after days of searching.
My second question concerns direct sequencing of pcr products. I´ve read in other posts about the reverse
reaction being better than the forward reaction. I observe the same phenomenon. Does anybody know the
reason? And i also observe something i haven`t read about yet: The signal of all bases is equally low, except for
the Cs in CpG dinucleotides that don`t seem to be converted (=methylated). I can`t explain why their signal is
more than three times higher than the signal of adjacent bases. I´m not experienced in sequencing, but could
it result from the high C noise and be a "calculated artifact"?
I´m looking forward to your answers,
Anne sorry for the long absence and late reply. for a positive control for your enrichment sequencing, without knowing your organism, you could try a centromeric or repeat element sequence? This maybe messy as you would also get signal in your input fractions because of the relative copy number. Another, if your organism is female, sequences on the X chromosome may have a similar pattern as imprinted control regions if there is X-inactivation?
As for direct sequencing, I have put the forward and revers primer phenomenon down to how the polymerase behaves differently when incorporating particular nucleotides, in the forward direction you are mainly incorporating TAG nucleotides, but in the reverse it's TAC. As for the spike in C's it's because the majority of your amplicon is TAG and as these are depleted during the sequencing reaction, there is an abundance of C's which give rise to the potential that more extensions are terminated at C leading to a higher signal.