Protocol Online logo
Top : Forum Archives: : DNA Methylation, Histone and Chromatin Study

pyrosequencing question - (May/03/2007 )

hello everyone,

I'm about to start a pyrosequencing project at the lab I'm working at and hopefully use those results to start writting my thesis tongue.gif . I've seen most of the pyrosequencing programs and results have been for mammalian studies, I'm working on plants instead, so there'll be a bit of difference in the sequence context, but that's not the problem. I have this doubt, I wonder if pyrosequencing will work well for DNA methylation quantification even if my sequences are not rich in CpG islands and be able to detect the differences in the methylation from each allele. The snp's are not very many but there are some I could use for designing the primers.

Thanks a lot for your help happy.gif

rodpck.

-rodpck-

I don't think that pyrosequencing will be able to differentiate between two separate alleles and tell you the methylation state for a particular chromosome. Pyrosequencing will tell you the relative abundance of a certain nucleotide in a certain position within your amplicon. So if you are trying to differentiate between two inherited alleles you (in theory) should see a 100:0, 50:50, or 0:100 ratio for a particular snp at that location.

To determine the methylation state of a particular allele, I think you should use the classic bisulfite PCR and cloning. That way in the sequence of the clone you will know which chromosome your amplicon was generated.

Somebody please correct me if I am wrong!

Thanks

Oh I see now, if you think that you can bias your amplicons based on a SNP then you should be able to do this, the # of CpG's is not so important, pyrosequencing will tell you the percent methylation for that amplicon. But, if I were on your committee I would grill you about how you know for certain that your primer was absolutely specific for one allele over another. IMHO I would still do the colony based analysis so you can see the SNP's.

-bunja-

QUOTE (bunja @ May 9 2007, 04:24 PM)
I don't think that pyrosequencing will be able to differentiate between two separate alleles and tell you the methylation state for a particular chromosome. Pyrosequencing will tell you the relative abundance of a certain nucleotide in a certain position within your amplicon. So if you are trying to differentiate between two inherited alleles you (in theory) should see a 100:0, 50:50, or 0:100 ratio for a particular snp at that location.

To determine the methylation state of a particular allele, I think you should use the classic bisulfite PCR and cloning. That way in the sequence of the clone you will know which chromosome your amplicon was generated.

Somebody please correct me if I am wrong!

Thanks

Oh I see now, if you think that you can bias your amplicons based on a SNP then you should be able to do this, the # of CpG's is not so important, pyrosequencing will tell you the percent methylation for that amplicon. But, if I were on your committee I would grill you about how you know for certain that your primer was absolutely specific for one allele over another. IMHO I would still do the colony based analysis so you can see the SNP's.


hi bunja,

thanks for your reply. certainly it would be difficult to determine that the amplicon would be 100% from the allele in matter, however that's the best option we've got so far.

we've done reverse transcriptase pcr and bisulfite pcr on these sequences, after analyzing the results i wanted to see if the transcription of these fragments comes only from 1 of the alleles since the plants i'm working with are controlled pollinations from 2 different inbreds.

i'll keep reading more to check if this is possible or i'm wrong. thanks again!

-rodpck-