Inconsistent bisulfite PCR - (Nov/29/2007 )

Sorry it would be a bit long.
Here is my problem.

I am trying to do bisulfite sequencing for the promoter region of a gene. The forward and reverse primer amplifies a 318bp region. I used cell line and brain gDNA as template. The PCR conditions were optimized three months ago. Both touchdown and standrad PCR worked fine (single band for bisulfite converted gDNA as template, no band/smear for unconverted gDNA). The sequence was found to be correct.

I am now repeating the experiment but the PCR is no longer working. I performed touchdown PCR using cell line and tumour and normal brain gDNA as template. Unfortunately, a faint band of ~320bp was obtained for unconverted gDNA. So probably if I clone and sequence the PCR product (using converted gDNA as template), I may also obtain sequences from unconverted template. I determined the optimal annealing temperature again by temperature gradient, and was found to be consistent with before. So I repeated the PCR but got more terrible results -- non specific band was found even in converted gDNA and there were multiple non-specific bands for unconverted gDNA (again, one of the bands has ~320bp).

My supervisor suspected I had contaminated the unconverted gDNA, so a faint band was obtained for this control. I changed all equipment (pipettes, tips, tubes) and reagents but the faint band was still there. What is more puzzling is that inconsistent results were found -- sometimes the non-specific bands for the unconverted gDNA control disappear completely, sometimes no band was obtained for all reactions in a PCR.

There is probably something wrong with the PCR conditions, but no matter what I changed (annealing temperature/time, extension time, no. of cycles, amount of gDNA), the results remained inconsistent. I extracted gDNA from cell line and confirmed the DNA is intact and free of RNA by gel electrophoresis and 260/280 was ~1.8, bisulfite conversion was done using this gDNA. But PCR results are still inconsistent.

I was wondering if I will get any unconverted sequence because the conversion rate was found to be 99-100% previously. But my supervisor suggests that if the PCR is optimized, the target band should not be found in unconverted gDNA control even if it is a waek band. So there is no way to do sequencing if I cannot get a good PCR result just like before.

Do you have any ideas how I could carry on?
Thank you very much.

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smells awfully like some kind of PCR amplification contamination, could your primer stocks be contaminated/degraded? have you tried replacing those?

N

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contamination is a really difficult thing to get rid of,

have you tried on top of replacing all your reagents, using someone else's set of pipettes as well as filter tips?

you could also try setting up your PCR in someone else's lab that you don't normally set up your PCR's in.

do you have dedicated pre- and post-pcr setup locations in your lab? could be something to think about.

Nick

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Thank you Nick, I used laminar flow hood for my PCR setup with regular UV irradiation, I tried wiping the hood and racks with 10% bleach solution before. I used another set of pipette that has not handled the target amplicon or template DNA and replaced all the reagents, from H2O to Taq, same result was obtained. I have extracted the gDNA and modified it again, I repeated the PCR for for 4 times and the 318bp band in the unmodified DNA is now gone. So I suspected that the unconverted gDNA used previously did have some problems (e.g. contamination with PCR product or poor quality due to presence of RNA).

But out of the four repeats, I still got one complete PCR failure with no band in almost all reactions, except a very faint band in 2 of the tubes. So this time it may not be DNA contamination or DNA quality problem but other things. My supervisor suspected that my modified DNA may be degraded becuase I have thawed it 3 or 4 times, I am not sure about this. I think the chance of setting up the PCR incorrectly or problematic reagents is low, what else could be wrong?

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hmmm most perplexing!!

how did you convert your DNA? with a kit or homebrew?

Nick

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Yes, this problem has persisted for a month and my supervisor is going to fire me soon,
I use Epigentek methylamp one step modification kit, it worked without problem 3 months ago.
And I used freshly prepared converted gDNA for my PCRs, usually I used up the DNA within a week.

So it seems there are only two possible causes, either I made mistakes in my PCR set up intermittently or there is something wrong with the PCR machine.

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Using a different PCR machine would solve this issue, which I think you have tried?

It could also be the stability of the converted DNA after this kit treatment. I am not familiar with the kit, but it could also be that your DNA has degraded and that is why you are getting no amplification in your samples.

Nick

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There are several occasions when I got good amplication in some tubes but not in a particular tube using another PCR machine so it could be a DNA problem. But the problem is I am still able to get amplification using the same modified DNA in subsequent PCRs, I wonder why the DNA has degraded in some tubes but not in others and in one time but not another. This really drives me crazy. Thank you for your suggestions.

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