Bisulfite sequencing PCR not working - (Apr/29/2010 )
Firstly, I find this bioforum a lifesaver! So I'm here again with some basic questions....
I have been doing MSP for the last while and the PCRs are working great for these. I have recently started looking at bisulfite sequencing PCR. I used methylprimer express to design my BSP primers and my bisulfite treated gDNA should be fine as it has been working well for all my MSPs but yet I am not getting any PCR product using my BSP primers. I started with temperature graded PCR and in a range of 50-62oC I am still not getting any product. My cycling conditions are: 95oC 30 secs, annealing temp 30 secs, 72oC 30 secs repeated 35 cycles followed by an extension at 72oC for 10 mins.
After reading the forums I can see that there are lots of conditions that can be changed on the cycling conditions, such as hot start PCR, increase number of cycles, nested PCR, lower extension temp etc. I am wondering from anyone that has a lot of experience with bisulfite sequencing PCR what conditions generally work well?? Also, do you need a larger amount of template DNA for bisulfite sequencing PCRs compared to MSPs? Can these products be directly sequenced using the same primers used to amplify the PCR product? Based on the companies I've spoken to that regularly sequence DNA they can, but I'd like to hear what you guys think on this
Any help on this topic would be much appreciated,
What is the size of your amplicon (is it AT rich?) And do you see huge primer dimmers in your gel?
epicrazy on May 4 2010, 05:48 AM said:
Thanks for your reply. I am looking at a number of genes and most product sizes are between 300-450 bp for bisulfite sequencing. I used methylprimer express to design the primers. The sequences are pretty AT rich after bisulfite treatment and I have had to use degenerate/wobble primers as I had no options with my sequences to avoid a CpG dinucleotide. The primer dimers are not that big at all - although they do vary depending on the temperature (I do temperature graded PCR to check primers first). Any ideas??
Also, since I posted this I did a nested PCR and got a product at one of my temperatures on the 2nd round of PCR.... But if I could optimise the conditions to amplify a product on the first round that would be great..
Great! I had a primer pair once which refused to amplify the first round as well, I am guessing it was because my primers were long about 30bp and the PCR efficiency was just not good enough; so designed a new one to about 22bp and it worked pretty well after that
epicrazy on May 6 2010, 03:16 AM said:
I can not get sequence from my treated Samples, my control DNA sample work as well but no any result (I mean sequencing result) from Bisulfite treated samples, If any body has any idea?
A cycling profile that worked for me was to have the first five cycles at 2C below the annealing temp and the following 35 cycles at the annealing temp.
Maybe if you drop your annealling temp a little you may get a bit more success.