In my experiment, did bisulfite sequencing of 195bp amplicon that contains 5 CpG island. We got methylation signal in all of these cpg island in controls and cases. This increases our doubt that will it could be attributed to inefficient conversion of DNA. Besides, we did not find any other C residues in our amplicon (May be an indicator of complete conversion). So, by this I need suggestions to confirm that this may not be a experiment artifact.
Dear Srinivasan, can you give more details on your experiment, especially your method of analysis of the methylation status?
You may also consider:
1. Checking the samples and controls to make sure they are the right ones, and then re-optimizing your conditions.
2. I assume you are sequencing your amplicon. If so, what Taq polymerase are you using? Low fidelity Taq may affect your sequencing, so try a HiFi Taq.
3. If you do direct sequencing, you may wish to try cloning your amplicon to increase reproducibility of your results.
Hope this helps.
Thanks for your comments Mr. Imam
1st point: Yes of-course we are comparing the results with controls, however in controls also we found methylation of all 5 cpg island.
2. The taq polymerase that I use is routinely used in sequencing of normal DNA samples in which we have never encountered such errors. Because when ever a mutation is identified we used to reconfirm that through reverse strand sequencing and other methods too.
3. I repeated those samples multiple time however the results are reproducible.
Dr. Matthias, CpG islands are not a novel one, these islands are already reported on literature. And they had shown some variations in methylation pattern between cases and controls.
Then to use some control, I should design a new set of primer, flanking region should be devoid of CpG islands. However, I already mentioned that only at those 5 cpg island I get signal for C nucleotide but rest of the C residues are completely converted.
- Badges
- Science method