Bisulfite conversion conundrum - (Aug/09/2012 )
Hello all! Have loved reading this forum, always so helpful.
I have recently joined a lab and am picking up a former member's dna methylation project. The previous member successfully amplified DNA from tissue, bisulfite converted and PCR amplified regions of interest. These regions were then sequenced. My job is to go back and confirm her results by hand before we can publish. Easy right? Wrong!
I cannot get her primers to work! I am isolating DNA from the same tissue. I am using the same kit to bisulfite convert (Zymo methyl gold). Same primers, same taq polymerase, same PCR cocktail. I've been running her primers on DNA from tissue (of "unknown" methylation status) and on DNA from the tissue that I have treated with M.SssI to fully methylate. I am sometimes able to get the correct size band, but only in the fully methylated DNA, never from the DNA from that tissue that has not been treated with M.SssI.
Now then, my helpful colleagues... where should I assume my hang up is? I know the primers "work," since the previous person was able to get a product from bisulfite DNA of unknown methylation status. I am thinking that perhaps my bisulfite conversion is not working efficiently, is that most likely? (Since the fully methylated DNA would not require as much "conversion power" from the reaction to convert the DNA?) Or is it possible the the M.SssI treatment and/or NEB buffer that I'm using is doing something to that DNA to increase the efficiency of the conversion reaction? Any and all ideas will be welcomed!
Hi texasepigenetics, welcome to bioforum and to the methylation field!
First of all, please be aware that bisulfite PCR is much more difficult than regular PCR due to degraded DNA after modification and many constraints on primers.
Have you followed the same PCR protocol the other member used such as cycle number, Ta, etc? If yes, the problem may lie in your DNA. The Zymo kit should be reliable. A big problem with bisulfite conversion is not the issue of incomplete conversion but recovery of DNA.
Can you post your pcr conditions here? Often you need two rounds of PCR to see bands.
Hi pcrman, thanks for the reply!
I am using the same PCR protocol as the other member- however she used a different thermocycler the Ta at which my methylated DNA produced a band was a few degrees higher than what she says she produced a band at using those primers. The PCR master mix I use has a aptamer inhibitor, TQ21-11. I think I've seen it discussed in these forums, also. I am using a basic non-proofreading taq polymerase.
I am doing two rounds:
PCR1 mix recipe:
MM2/TQ21 mastermix 5x 20%
Taq polymerase 5U/ul 1%
Mix at room temperature. Let the oligo bind to the enzyme.
Water 77%
Mix, transfer on ice and keep on ice from now on.
Primer forward 10 uM 1% final 0.1 uM
Primer reverse 10 uM 1%
Aliquot in PCR tubes or wells (18 ul per well). Keep on ice.
Add bisulfite-treated DNA 2 uL
Heat up the PCR block to 95C.
Transfer the PCR tubes or plate from ice to the hot block.
Start cycling:
Initial denaturation 95C 5 minutes.
Then cycle denaturation 94C 15 seconds, annealing/extension 60 seconds at 60C. Use 40 cycles.
If your annealing temperature needs to be below 60C, add an extension step of 72C for 10 seconds. (mine are, so I added it)
PCR2 mix recipe:
MM2 mastermix 5x 20%
Taq polymerase 5U/ul 1%
Water 77%
Mix, transfer on ice and keep on ice from now on.
Primer forward (nested)10 uM 1% final 0.1 uM
Primer reverse 10 uM 1%
Aliquot in PCR tubes or wells (24.5 ul per well). Keep on ice.
Add 0.5uL PCR1.
Heat up the PCR block to 95C.
Transfer the PCR tubes or plate from ice to the hot block.
Start cycling:
Initial denaturation 95C 5 minutes.
Then cycle denaturation 94C 15 seconds, annealing/extension 60 seconds at 60C. Use 40 cycles.
If your annealing temperature needs to be below 60C, add an extension step of 72C for 10 seconds (mine are, so I added it).
For recovery of my DNA, I am using the columns provided with the zymo kit, but have been eluting with water instead of the included elution buffer- could that be the problem? I don't think I've ever eluted with an elution buffer that comes with columns, in case the components inhibit downstream reactions...
Your PCR conditions appear OK to me. The only thing the concerns me is the 60C Ta which is a bit too high for BSP.
If you use the same primers left from your colleague, there is also a concern of primer degradation.
A possible explanation for successful amplification of
Sorry, I should have corrected the annealing temp- I performed a gradient PCR between 60 and 50- the annealing temp for the primers that did work on the methylated DNA ended up being around 54*C.
I purchased a new stock of primers, not using her old ones.
I am also thinking that it may be as you suggested- that M.SssI is causing additional denaturing or fragmentation. Maybe I'll throw my DNA into the bioruptor for a quick round of sonication before my next trial...
I should also mention that this region is a CpG island- is it also possible that because the region is hypomethylated, that there are so many uracils in the sequence following conversion that it is degrading?
Finally, anyone have insight into using water rather than the elution buffer provided with the zymo gold methylation kit?
Thanks!!
An update for anyone interested: after working with someone with lots more methylation analysis than myself, it seems the primers I was using are poorly designed. I'll be redesigning following my experienced friend's guidelines and will update if I have any success with what I did for the redesign.
But you said your former colleague got good results using the same primers. Anyway, please keep us posted, and good luck!
Yes, I was under the impression her results were "good," however it seems that needing performing the PCR 10 plus times before you get a band at the proper size does not constitute a good bisulfite-PCR 
Oops!
texasepigenetics-
Were you able to re-design good primers based on your colleague's guidelines? It would be great to hear your tips!