There can be different reasons which could have caused the BS-PCR to not work, for now, what I can think of include:
1) The annealing temperature setting in the thermocycler: the Tm of my Forward primer is 48C and the Tm of my Reverse primer is 66C, the difference in the temperature between the pair of primers is sort of large (18C), could this be a problem? if not, then what temperature would you suggest I use? I used 48C for my previous run.
2) There could be some issues with the bisulfite conversion step: I have not done any thing to check if the conversion was complete. And one of my lab mates told me that if the bisulfite treat was too much, then it could do damage to the DNA, and I did not know anyway to check that either.
3) I used MethylPrimer express to design my BS-PCR primers: It is the first time I design primers (again, I had no wet lab experience before). I did not know if there is anything I need to pay attention to when designing primers, especially for BS-PCR primers.
I appreciate any of your insights here.
You have not picked an easy entry point into the world of the wet lab! Designing primers for bisulfite-specific PCR and/or methyl-specific PCR is challenging! I would keep amplicon size at less than 250 bp if appropriate for your application. Here are some suggestions for tools for bisulfite PCR primer design & visualization:
Web sites
Methprimer (converts sequence, designs primers, displays alignment, identifies CpG islands) http://www.urogene.org/methprimer/index1.html
BiSearch Primer Design and Search tool (ePCR, primer specificity screening) http://bisearch.enzim.hu/
Methgraph (aligns amplicons with Human genome browser http://genome.ucsc.edu/] features) http://mellfire.ugent.be/public/methgraph/visualizer.php
Primer 3 (as Nadine suggested) is good for primer design, but you will have to manually transform your sequences to bisulfite converted forms (methylated AND unmethylated versions!)
Programs
Methyl Primer Express (Applied Biosystems)
Useful representations for “inspection”
Generate methylated=>bisulfite, unmethylated=>bisulfite sequences
Align with each other and with original genomic to find primers that will amplify bisulfite treated methylated and unmethylated forms--but not original genomic.
Design new primers. 18C is WAY to big of a gap. Should be no more than 5C difference. $8C is too low too, so try and make the Tm higher
OK, 48 and 66. There's your problem. Your annealing temperature should be between 50-60C and ideally both primers should have near identical Tms. If not, no more than 2C difference between the two, even then I consider that being a potential problem. Let us know what your PCR temp profile is.
Hi, maybe you can design your primer with this free software. It's pretty easy to use it. http://frodo.wi.mit.edu/primer3/
How large is your amplicon? BS-PCR should be less than 900bp. Maybe that's also a problem..
Best, Nadine
You have not picked an easy entry point into the world of the wet lab! Designing primers for bisulfite-specific PCR and/or methyl-specific PCR is challenging! I would keep amplicon size at less than 250 bp if appropriate for your application. Here are some suggestions for tools for bisulfite PCR primer design & visualization:
Web sites
Methprimer (converts sequence, designs primers, displays alignment, identifies CpG islands) http://www.urogene.org/methprimer/index1.html
BiSearch Primer Design and Search tool (ePCR, primer specificity screening) http://bisearch.enzim.hu/
Methgraph (aligns amplicons with Human genome browser http://genome.ucsc.edu/] features) http://mellfire.ugent.be/public/methgraph/visualizer.php
Primer 3 (as Nadine suggested) is good for primer design, but you will have to manually transform your sequences to bisulfite converted forms (methylated AND unmethylated versions!)
Programs
Methyl Primer Express (Applied Biosystems)
Useful representations for “inspection”
Generate methylated=>bisulfite, unmethylated=>bisulfite sequences
Align with each other and with original genomic to find primers that will amplify bisulfite treated methylated and unmethylated forms--but not original genomic.
Bisulfite conversion is also a VERY tricky business! I would suggest you have a look at using some bisulfite treatment controls...I know Qiagen has a kit with 3 control DNA-types to check conversion. You don't mention whether you do the conversion with a kit? The kits are usually easy to use, but needs some changes sometimes to ensure you don't lose to much of you sample...and it needs alot of practice to ensure full conversion.
Good luck!!
Thank all you guys for the comments/suggestions!
@David Barker: the total length of my amplicon is about 670bp. In order to cover all the 7 CpG sites in the fragment, I designed two pairs of primers, so the amplicon is split to two smaller pieces, one piece is of 268bp, and the other is of 308 bp. As you said, it is better to keep the the size at less than 250 pb, so maybe one thing I can try is to cut the whole amplicon to three pieces and keep each piece of under 250bp.
As for the primer design, I am using Methyl Primer Express for now. I would like to try the other websites/software you recommend to see what the differences are like.
Thank you!
@ Stephen Doyle: Yeah, I was concerned about the 18C temperature gap, too, but my fellow students told me it was quite common in BS-PCR primer design. The reason is that, I added a 'universal biotin tag' to the 5' end of the reverse enzyme, which makes the 'new reverse enzyme' 21 bp longer than the Forward primer. The Tm for the forward primer is 48C and the Tm for the Reverse enzyme is 66C. Maybe I should try a higher Tm, say, 51 or 53, as you suggested?
Thank you!
@ John Mclntyre: As I just mentioned above, The Tm for Reverse Primer is 48C, and Tm for Forward primer is 66C. My PCR profile is:
------------------------
95C 15min
45 cycles of
94C 30s
45C 30s
60C 30s
60C 10min
4C forever
----------------------------
Thank you!
@Nadine A. Gund: I would love to try the software that you recommend. The total size of my amplicon is ~670bp, and I split it to two smaller pieces: one is of 268bp, the other is of 308bp.
Thank you
@Chrisna Gouws: yes, we use Qiagen kit for the bisulfite conversion but I don't quite remember exactly which kit we used, I will double check. I totally agree with you about the fact that BS-conversion is tricky! too much treatment, the DNA could be damaged, not enought treatment, the conversion would not be complete. At this point, I have not figured a way to check how good our BS-conversion step was. Maybe I should try the kit that you mentioned to check the quality of the BS-treated DNA. This is a very important step.
Thank you!
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