1. I am new to studies on DNA methylation, and I understand that when using BS-converted DNA for any downstream application, it is crucial to correctly quantify the mostly degraded DNA. It appears that pre-pcr, i get fairly reliable nanodrop readings when I quantify BS-converted DNA using factor 40 similar to RNA (got this suggestion off the Zymokit manual). After PCR, the results dont seem to be as reliable. Can I really measure BS-converted DNA post-pcr and after clean up, using nanodrop?
2. I have come across articles with gel pictures indicating complete conversion of DNA following BS-treatment with only a smear in each lane. What I dont understand is, shouldnt one be looking for the right-sized bands after running amplified BS-converted DNA? Or does it mean complete conversion indicated by a smear on a gel is checked for using the BS-converted DNA before pcr?
3. I have tried direct sequencing of my amplified BS-converted DNA on Beckman Coulter GeXP but i keep getting abrupt signal dropouts. this could be due to the GC-rich nature of the CpG island I am trying to sequence as I have been informed. So i tried using 5% DMSO in the PCR reaction. I am still optimizing the direct sequencing but what interests me is that despite reports that DMSO doesnt interfere with DNA amplification, i consistently get fainter bands on gel electrophoresis suggesting less amplification of the DNA. Anyone has experience using DMSO in a pcr? Also, is direct sequencing of such products (without cloning) a good idea?
Any suggestions?
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
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