First of all this forum is a Godsend for me given that I've been banging my head on a wall trying to get my Bilsulfite PCRs to work for a month now with absolutely no luck. By that I mean I think I've gotten the bisulfite treatment part down but that my primer design for whatever reason is the cause of complete and utter failure. Please read and let me know what you think. I apologize that it's long but if I figure if I put it all out there it will help with troubleshooting. Thanks guys!
Here is my bisulfite treatment protocol:
- Either digest 4 ug of genomic DNA with a restriction enzyme that doesn't cut in my targeted CpG islands (I digest 4 ug because I lose 50% of this during column purification in preparation for the next step) OR run 2 ug DNA through a 26.5 gauge needle 5X to shear it.
- I UV spec DNA at this point just to make sure I have enough going into bisulfite treatment. I usually have 1.5 - 2 ug going forward. So far so good.
- Denature DNA in FRESH 0.3 M NaOH and incubate at 37C for 20 mins
- Add FRESH Bisulfite/Hydroquinone (final concs 2.4 M/0.5 mM respectively) and incubate at 50C for 16 hours wrapped in foil
- Column purify into 40 ul H2O (Promega Wizard)
- Add 4.5 ul of FRESH 3M NaOH to desulfonate at room temp for 5 mins.
- Ethanol precipitate with ammonium acetate (no glycogen) overnight. At this point I get good pellets so I figure I'm good so far. NOTE: I spin multiple times and pipette out the 100% EtOH. I do not wash with 70% EtOH before resuspension in 1X TE. Is this crucial?
- Resuspend in TE. Store at -20.
At this point I test for full conversion. I take my bisulfite treated DNA and UV spec it and it always has a positive reading (~30 ng/ul or almost 2 ug usually each time) with OD 260/280 readings from 1.2 - 1.5. Furthermore I've tried doing regular PCRs (amplicon size= 300 bp) using non-bisulfite PCR primers on bisulfite-treated DNA to see if I can amplify any residual unconverted DNA and in most cases it comes up negative (a few cases here and there are positive which means incomplete conversion). If my bisulfite-treated DNA passes these two checks then I assume it's good-to-go for bisulfite PCRs. Is this assumption correct? Should I be checking for something else?
Next is PCR time where usually a lot of cursing occurs. I've tried manually designing primers and using MethPrimer as a primer design tool. In all cases I have never gotten a single PCR band. I've tried about 10 different PCRs now and in a subset I've even tried hemi-nested and fully-nested PCRs and they've all failed to yield any bands. These amplicons are 300-500 bp long so I don't think I'm pushing the envelope too hard here. My standard thermocycler profile is as follows:
95C for 5 mins
95C for 30 sec
45C for 30 sec
72C for 3 mins
72C for 7 mins
I used to do 55 anneal but when I was getting no bands, I switched to 45C anneal. After this switch, I did nested and hemi-nested PCRs on a some samples but those didn't work either. In some cases I see some primer-dimers but most of the time there's absolutely nothing even when I run the whole PCR reaction out on a gel.
My PCR conditions (50 ul reactions) are as follows:
200 nM each primer
200 uM dNTPs each
3 mM MgCl in standard Roche 10X PCR buffer
2.5 U Platinum Taq (Invitrogen) -- hotstart and no proofreading (I think).
~100 ng of bisulfite-treated DNA in TE per reaction (usually ~3 ul)
In regards to primer deisgn, I've been very careful not to include any CpGs within my primer sequences. Just to give you an idea of some of the primers I've been working with:
I know some of my primers have long stretches of Ts which aren't a good thing but these tend to be the 5' portion of the primer and not the 3' half. At this point, I am at a loss as to where to go from here despite having read this forum religiously over the past day and a half. The biggest questions in my head:
Is my bisulfite treatment of DNA working? I think it is but maybe I'm missing something.
Should I have washed my pellet with 70% EtOH to desalt? I don't think this is that crucial, but maybe it is?
Are my primers poorly designed? I'm thinking yes and this is probably the cause of my failure, but I have no way to confirm since my 'positive control' primers (i.e. those taken from published papers run under their published conditions) have also failed. Maybe my PCR conditions aren't optimal. Major ARGH.
The only things I can think of at this point are to try a new bisulfite treatment method (agarose beads) or maybe try Methyl Primer Express as a primer design tool, but I suspect it's something other than that. Any suggestions as to where to go from here would be appreciated. As Tiger Woods would say, "Quickly. Huge. Bye."
Edited by gradeachouster, 10 December 2009 - 11:18 PM.