Dear mustapha

I think you should optimize tempet DNA with DNA methylated as a control. there

are two presumption,1-noncompletly converting that be checking by control DNA methylated cause to smear 2- Over converting that cause to degradation of DNA and check with control DNA methylated and use two volume of templet.

I thinke your concentration of DMSO is higher and 1% is bether than.

finally , you should be cloning your product in vector for sequencing because there are mix of un/methtetion of DNA.

good luck

Thanks Mohammad. I was just curious to know whether smearing could be used as an indicator of successful conversion, but now I understand it is indicative of a problem with my BS-conversion.

As for the DMSO concentration, I have seen 5% reported in many literature but not 1%. Any reference for the 1%?

btw, below is a feedback to the 3 questions above, from another forum. Could be of interest to some:

1. Post PCR it should be double stranded again. So yes indeed. Be aware that you do not have to convert to RNA since the "U" have been converted by the polymerase to regular DNA base again.

2. I havent read any such articles, a smear is never a good indication of bisulfite conversion. We and all other DNA meth. labs I know use Sanger Sequencing of converted DNA to get an idea of conversion.

When testing BS-PCR primers: always incorporate a few reactions on gDNA, these should be blanco (otherwise your DNA meth quantification is dependent on your bisulfite conversion --> unreliable).

Your product should also be unique. Its PCR... a-specific bands are a big no-no. No unique bands? Redo PCR optimalization and if that does not work redesign primers.

3. Please also be aware that most software used to watch your traces inflate the C background signal; depending how the software works this might also influence the other 3 bases calibration and signal intensities.

I always look at the raw signal from the ABI. It might help to add a bit more T to your dNTP mix. It might run out rapidly depending on the amount of starting material in your sanger reaction.

About DMSO, no idea, sorry.

I think that the smear of DNA after bisulfite is quite normal. The treatment is highly "toxic" for the DNA integrity. It is the reason we do not perform PCR with large fragment. The regular size of PCR fragment are around 150-350bp. When you perform PCR, you need to clone your fragment before the sequencing and sequence more than one clone. The clone number depend of the sensitivity you would like to have (10 clone or more).

for DMSO, I agree, no more than 1%. You could try betaine. But for bisulfite, often, you need to increase the amount of template and use an high fidelity Taq pol due to the low quality of BS template.

finally able to sequence my fragment without cloning. the issue with my reactions was apparently the low fidelity Taq i was using. Anyway, I guess Stepane is right with the cloning part, to ensure reproducibility. Will try that as well. Thanks Mohammad and Stephane

Hi Mustapha, 

I noted that you have managed to sequence your fragments without cloning. I am also trying to do the same. However, I have having problems with the baseline noise of my sequencing trace. Did you have any such issue? How did you overcome it?