Dear Researches,
At the moment I’m working on one PSQ assay and I have some difficulties. In brief, I have 2 Sequencing primers (S1 and S2) for the same PCR product: S1 = reads a sequence of 36 bp; S2 = reads a sequence of 62 bp.
Both Sequencing and PCR primers were taken from literature and were used successfully by others to study DNA methylation.
Now, for my analysis I used the same PCR primers and S2 primer. After PCR, I checked the samples to see if I have any contamination, but I had one clear band.
However, all samples did not work properly on PSQ, almost all of them were “red”, including commercial 100% methylated DNA, that I used as an additional control. (errors given by PSQ: Uncertainty due to high peak height deviation at dispensation ... or bisulfite conversion problems).
Next, I re-ordered all primers and was using new reagents. In addition, I used samples from 3 different bisulfite conversions (2 sample sets were used for other studies, they worked well and were stored at -80) and 100% methylated DNA. Again, samples did not work properly with S2 primer.
Then, I run two more experiments with the same samples (3 different bisulfite conversions + 100% meth.DNA):
1st set: with S1 primer (using the same PCR primers as for S2); 2nd set: with PCR and Sequencing primers for other gene.
Result = all samples worked well !
As far as I know 62 bp should work quite well on PSQ, so I'm not sure if length can be the main problem. In addition, I'm always careful with Enzyme and Substrate, so they should work properly.
Can you please let me know if you have any ideas why S2 does not work, but S1 does work well?
In case you had a similar experience with PSQ, can you please share how did you solved this problem.
Thank you in advance !
this link useful
https://currentprotocols.onlinelibrary.wiley.com/doi/pdf/10.1002/0471142727.mb0715s104
regards
Hi
as far I know the ideal primer length for pyrosequencing should be around 18-24 bp. bcoz your s1 is smaller compared to s2 its working. try designing a smaller length primer, may work. good luck
Dear Bhagyalakshmi,
Thank you, but I need to mention that:
36 bp and 62 bp are not the primers length, but the length of a DNA sequences that are read by sequencing primers S2 and S1.
The length of primers S1 and S2 is 18 and 21 bp (I didn’t mentioned it in my description).
Dear all,
Just a little update.
Few weeks ago I found a solution for my issue, so the case is solved.
A very short description of how I have done it:
1) I have designed different sequencing primers that read the same DNA region that I’m interested in. The primers differed by length (the shortest was 14 bp, longest 25 bp) and of course by sequence.
2) I run each primer on 2 different samples and I made 2 types of assay for each primer (1st assay for a long sequence and 2nd assays for a short sequence).
Result: I found a few sequencing primers that read a long DNA sequence very well (without errors) on Pyrosequensing.
Conclusions:
The sequencing primers from the literature might not always work very well and you might need to adapt them a bit.
Why it might happen? Well, in brief, it might be related to different PSQ machines; updates in the software that is used to design an assay and analyze the results; and reagents (for example the type of cartridge that is used). Of course there might be more reasons, these are the most obvious.
Hope this information will be useful for someone.
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