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Best technique to analyse methylation level of CpG island


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#1 GalkaPiteskaya

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Posted 30 July 2014 - 06:01 AM

I have used bisulfite sequensing to analyse methylation level of several CpG sites in a region of interest. Length of most of my amplicons did not exceed 400bp and they were not CpG site dense. But now I would like to analyse the methylation level of several CpG islands with length 800-2000 bp and very CpG sites dense. As I know CpG dense regions are quite difficult to amplify. So I do not know if it makes sence to try another technique which is based on bis-DNA amplification, melting curve assay for example. Which another approach might be used here?



#2 phage434

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Posted 30 July 2014 - 10:25 AM

I've never had an especially difficult time with 800 bp bisulfite PCR reactions. Even if you didn't want to do them, then 2-3 overlapping sequencing reactions would solve your problem.



#3 GalkaPiteskaya

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Posted 31 July 2014 - 01:00 AM

Thank you! You never had problems even if it was CpG rich regions?



#4 phage434

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Posted 31 July 2014 - 04:28 AM

PCR of bisulfite converted DNA is more difficult in general. You can use nested primers, which helps a lot with specificity. Another issue that arises is that converted DNA is dominated by AT rich regions. You have converted most of the C's to T's, so the GC content has roughly been halved. With the G vs. C strand bias in most genomes, this an be even more extreme in one direction vs. another. When ampllifying very low GC regions, it can be important to lower the extension temperature (not the annealing temperature). During extension, if the 3' end of the extending fragment has extremely low GC (a stretch of 30 bp or so of GC < 2-3 bases) then the strand will not bind to its complement, and the PCR enzyme will fail to extend. This can usually be solved by lowering the extension temperature to 63-65 rather than 72 (and lengthening the extension time). CpG islands actually make this less of a problem, since they still have many C's. But all it takes to prevent normal temperature PCR extension is one 30 bp region with very low GC.






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