Bisulfite treated DNA specific primer set critique? - (Aug/18/2005 )

Hey nick,

Can I get your opinion on this primer set? I designed them with the help of perl primer but I am not sure if I included an optimal number of conversion events to select for the converted template.

No 3' dimers detected by perl primer but how worried should I be about the non-extensible primer dimer @ 37C?

-Cw

Hi cwong,

the primer pair looks fine, it will pay to select a third primer so you can perform a hemi-nested PCR to ensure you obtain enough product.

With the non-extensible primer dimer, you do not have to worry about that. It has been defaulted to be 37C, you can change this in the preferences (the spanner button) to the Tm of your primer and it will go away (ie: there will be none at the temperature of your Tm). I should mention this to the author.

Good luck with it!

Nick

Hmm ok, so I tested these new primers on my bisulfite treated DNA and didn't get a product band, but got bands <100 bp which I assume are primer dimers. I also am getting a band in my no template control and no band in my no taq control. And no I did not run a hotstart reaction.

So I am a little confused as to what I can do, my annealing temperature is set to only 2 degrees below the perl primer calculated Tm. Would it be wise to bump my annealing to my Tm or higher?

Quick off topic question:

Is the reason why bisulfite treated genomic DNA can't be visualized on a gel because it is highly denatured and single stranded? My labmates and I are having a discussion about it.

-CW

CW,

I would suggest a hotstart PCR, A manual hotstart will suffice (wait for the thermocycler to reach denaturation temperature before placing your tubes in.

2 deg below the calculated Tm is fine, what you are seeing is primer dimer. You could also increase the amount of bisulfite DNA in your reaction (place 2uL instead of the one).

As for bisulfite DNA in general, indeed, treated DNA becomes single stranded in part because the strands become non-complimentary after treatment, futhermore if you are using conventional methods, the bisulfite has a tendancy to degrade your DNA so you start off with 2ug of DNA you will probably get back 200ng in total although exact measurments have not been published.

Nick

In my experience the best way to get good products and negative controls, with minmal cycle number, is to use a good hot start taq polymerase. I would personally recomend BD biosciences Platinum Taq, it's expensive but has not failed me...yet.

Ok so I ran a hotstart pcr on my samples using the primer set described above and a magnesium titration from 2-7mM in 1 mM increments with my annealing temp set a couple of degrees below primer perl primer calculated Tm. No product .

Has anyone had luck using NEB DNA taq polymerase/Thermopol buffer in amplifying bisulfite treated DNA? Its what we had in the lab so I figured it was worth a shot, but even with a manual hotstart (cycler to 94C, added samples minus taq for 2 minutes to heat them up, added taq directly to tubes) I'm still not getting a product.

I don't know what to make of this either, the primer dimers which I saw earlier are still there, albeit much fainter (barely visible after ethidium bromide staining) in all my samples EXCEPT my no template control in which the band was as strong as before. minus taq control still showing as negative so I don't think it is water contamination.

I'm thinking of trying a touchdown PCR, but if its actually my polymerase thats acting up then I don't know if it would be worth the effort. So what do you all think? Should I redesign my primers, buy a new polymerase, or touchdown PCR to try different annealing temps??

-CW

hi cwong,

how did you prepare you bisulfite DNA? with a kit or the conventional method?

you could increase the amount to template you are using to 2ul and see how this goes.

are you performing a heminested PCR (ie: two rounds of PCR?)

if you are only performing 1 (30 cycles) it may not be enough to visualise on a gel and usually requires a second PCR step with a nested or heminested Primer set.

you could try a touch down and this will remove your primer dimer.

Our group routinely use Promega master mix taq, I have also used Sigma Taq with no problems at all. I performed a psuedo-manual hotstart place the PCR tubes with reaction and enzyme when the PCR machine has reached 96C. heatign your reaction to that temp and then adding the enzyme will increase the risk of cross comtamination.

Good luck!

Nick

how did you prepare you bisulfite DNA? with a kit or the conventional method?


I've been using the modified bisulfite treatment protocol by olek (posted by Labtechie too) with the DNA embedded in LMP agar, since I had concerns about my DNA yields from small primary epithelial cells. Since I'm having real issues troubleshooting my problem right now I believe that I will switch to a more conventional method on cell line DNA and see how that goes. I've seen many bisulfite treatment protocols posted which all apparently use different molar concentrations of Bisulfite/Hydroquinone. Which do you use Nick? 3.8 M, 5M or 6.3M? Do you use NaOH or Ammonium acetate for desulfonation and at what concentration?


you could increase the amount to template you are using to 2ul and see how this goes.


I've tried doubling my template to no avail. No product is showing up.


are you performing a heminested PCR (ie: two rounds of PCR?)


No. I was not originally but have since switched and modified the above primer set to include a hemi-nested primer. 2nd round of amplification (30 cycles both runs) results in no product band, but 5 uL of product on a agarose gel is visualized as a smear which I would assume is from nonspecific amplification? No dimers though.



you could try a touch down and this will remove your primer dimer.


Will give this a shot. But if it doesn't work I believe I will start from the beginning, using more conventional methods than I've been trying. For touchdown, is starting @ 5C above Tm and decreasing 1C every 2 cycles till 2C below TM considered acceptable?

This is the modified primer set with the hemi nested primer included:

Hi cwong,

you primers seem fine by me!

you should give the conventional method a try as I don't think it is necessary to use LMP method.

I have been using a 3.6M bisulfite solution, essentially you want a saturated solution of bisulfite, I think with higher concentrations (higher than 3.6M) you would have quite a bit of undissolved bisulfite which is fine but I think a little wasteful. hydroquinone is used to stabilse the reaction and I use 100mM with no problem at all.

I also use ammonium acetate at 5M concentration for desulfonation, NaOH is used to denature DNA into single strands....(see attached document).

Your touchdown method also seems fine.


good luck!!!

Nick

Thanks for the protocol nick, very helpful. I'll give it a shot and post any positive results.