How to explain my MSP sequencing result? - (Jul/08/2005 )
Hey:
to determine the convert efficency of bisulfite modification,My MSP amplicon was sequenced.But the result was so confused.
In the amplicon of methylated primer,all non CpG "C" were converted to "T" perfectly,but CpG "C" were still "C". And in the amplicon of unmethylated primer, all non CpG "C" were converted to "T" and almost all CpG "C" were conveted "T" too.
There were many CpG sites in the region between MSP primer.I think maybe not all of these CpG sites methylated,meybe there were still some CpG "C" unmethylated. But in My amplicon of methylated primer,all CpG "C" were still "C". Can I believe all these CpG "C" methylated ? but WHY in the amplicon of unmethylated primer,almost all CpG "C" between primer set were conveted "T" ??? (Same templat was used in the PCR rection) and How to evaluate the convert efficency of My bisulfite modification???
The PCR product was conducted by direct sequencing, Not clone sequencing.
Hey:
I still have a question.Why we not sequence the MSP amplicon to learn more CpG "C" methylation condition?
When study promoter methylation by BSP with cancer tissure,because of contamination of normal tissue, direct sequencing almost can not work . BUT with MSP we can get methylated DNA molecule,and direct sequencing mybe can work.so we can get more CpG "C" methylation condition. Am I right?? 
In my research,templat was prepared with LCM.then BSP was conducted.can I use direct sequencing???
Hi rockysofar,
Your sequencing results are nothing out of expectation. First of all, sequencing MSP or USP products doesn't make any sense because these products are amplified with bias and that's why you found high level of methylation in MSP product and no methylation in USP product. Anyway, the sequencing results probably tell you that your PCR results were specific.
If you want to know the methylation status of all CpGs within the amplicon, use BSP primers instead.
Hey Pcrman
Thanks for reply!
But why MSP or USP primer amplify with bias? MSP and USP primer are different in CpG sites which located in the primer, but between the primers,there are more CpG sites maybe methylated differently. do you mean these CpG sites methylation condition affect PCR amplicon with MSP or USP primer?
AND if I prepare templat with laser capture microdissection,direct sequencing can be conducted to BSP product??
Hi rockysofar,
Your template is a mixture of methylated and unmethylated DNA molecules. MSP primers preferentially amplify methylated DNA (that is what you expect) thus their PCR product will show high level of methylation on sequencing. USP primers do the opposite.
If you can do MSP on microdissected DNA, you should also be able to do BSP. Usually two rounds of amplification are needed to get enough product for sequencing. You can either use nested primers or reamplify using the same set of primers.
Thank you very much!
Your suggestion is what I am going to do.I have design nested primer to amplify the gene promoter CpG island.
Thanks for reply!
But why MSP or USP primer amplify with bias? MSP and USP primer are different in CpG sites which located in the primer, but between the primers,there are more CpG sites maybe methylated differently. do you mean these CpG sites methylation condition affect PCR amplicon with MSP or USP primer?
AND if I prepare templat with laser capture microdissection,direct sequencing can be conducted to BSP product??
Hi rockysofar,
I intend to use LCM-extracted DNA for my MSP. However, my MSP was failed.
1. Did you quantify your LCM extracted DNA? How much did you get?
2. How did you fix your tissue? Using formalin?
3. My supervisor told me that it is very difficult to amplify LCM extracted DNA, especially if you want the size of more than 300 bp.
Thank you.