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PCR amplification before bisulfite conversion


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#1 wannea

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Posted 06 October 2010 - 02:04 PM

Hi everyone,

I have been working with DNA extracted from paraffin-embedded tissue and I want to do a methylation analysis of a promoter. The problem is that my genomic DNA is not as good as I would like so after bisulfite conversion (Qiagen EpiTech kit), when I try to do a BSP, I canít get any band.

Because of that I am thinking on doing the gene amplification before making the bisulfite conversion. Does anyone know if it is possible? Have anyone try it? Iím not sure if itís going to work because bisulfite conversion degrades the most part of DNA so itís more likely to do it with small DNA fragments. Any ideas?

Thanks!

#2 methylnick

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Posted 06 October 2010 - 02:11 PM

Pcr before conversion ERASES all DNA methylation marks. Do not do this!

#3 wannea

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Posted 07 October 2010 - 12:22 AM

Thank you very much for the answer, what can I do now? I have no bands in my BSP, I've tried everything, I did nested PCR, differents sample concentration, differents MgCl2 concentration, I've tried to increase the Tm in my PCR cicles, I've changed my Taq, I've read everything on this foro that could help but I still waiting for my bands. My primers works fine with other samples so I think they aren't the problem. I really don't know what to do.

#4 methylnick

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Posted 07 October 2010 - 02:34 AM

how big is the amplicon you want to amplify by PCR? for FFPE samples you want to be under 200bp because of the highly fragmented nature of your sample

#5 wannea

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Posted 07 October 2010 - 05:30 AM

only 200bp??? my promoter is around 300bp, is it mean that I'm not going to amplify nothing never???

and another question is if I try to amplify the same promotor of a cell line instead of FFPE samples how big is the amplicon I can amplify?? can I try to amplify maybe 1000bp?

#6 ElHo

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Posted 13 October 2010 - 01:31 AM

If DNA was isolated from a cell line, amplicons up to 1000 bps should pose no problems. This is a good way to check whether your problem lies with the fragmented nature of your FFPE DNA or with the BSP. So I suggest to establish your protocol on cell line DNA and if it works, switch to FFPE DNA.

#7 wannea

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Posted 18 October 2010 - 12:14 AM

Ok, thank you very much, i'll try this week




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