4 replies to this topic

[Michaelro]

Hi everyone
I have a problem and I'd like to hear your opinion
I made pyrosequencing on the same SNP once on the normal genomic template and once on the same template treated with bisulfite.
According to the pyrosequencing results, on non-treated template, allelic frequency of this particular SNP was similar to the frequency obtained from other sources like Illumina and microarray.
However, I obtained significantly different allelic frequency for the same SNP on Bis treated template.
Since the SNP is C/T - I performed the PCR on the complementary strand (A/G polymorphism which is supposed not to be changed following Bis conversion).
It seems like unbalanced allelic amplification but is it something characteristic to PCR on Bis treated templates.
In adition, we are not talking about CpG island but short amplicon with 3 CpGs inside.
I'm using touchdown PCR for bisulfite and regular PCR on normal template.
Any suggestions and ideas will be wellcome
Thanks
Michael

[pcrman]

What is the base following the SNP? If it is a G, there C allele will form with the following G a CpG site which is prone to methylation. If the C is not methylated, after bisulfite treatment, the C will appear as T so you will get high allelic freq for T.  Hope I have understood your question correctly.

[Michaelro]

pcrman, on Aug 1 2009, 09:40 AM, said:

What is the base following the SNP? If it is a G, there C allele will form with the following G a CpG site which is prone to methylation. If the C is not methylated, after bisulfite treatment, the C will appear as T so you will get high allelic freq for T.  Hope I have understood your question correctly.

Thank You very much
The bisulfite PCR as designed in the way that only the strand uneffected by bisulfite conversion should be amplified (if the SNP is C/T - I amplify the complementary strand)
The actual reason was the sequencing primer design which placed the SNP too far from the sequencing primer.
After the re-design using the pyrosequencer software, I've obtained the expected results.
Best
Michael

[Michaelro]

pcrman, on Aug 1 2009, 09:40 AM, said:

What is the base following the SNP? If it is a G, there C allele will form with the following G a CpG site which is prone to methylation. If the C is not methylated, after bisulfite treatment, the C will appear as T so you will get high allelic freq for T.  Hope I have understood your question correctly.

Furthermore, if the SNP is part of the CpG or creates the CpG - I allways take this into consideration.

[methylnick]

Bisulftie is strand specific. Are you looking at the correct strand? I always get this wrong with my work