Different efficiencies for fully methylated and unmethylated

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Different efficiencies for fully methylated and unmethylated

Postby eyes » Mar 15 2017 9:38 am

Hallo guys,

I am studying a genomic locus in some tumoral specimens with the aim of clarifying if there are differences in the methylation status of about 30 CpGs among different samples (two groups of samples differing for the expression pattern of a specific gene).

I designed primers following the most common recommendations (e.g. amplicon not longer than 350 nt, primer with a CpG at the 5' end) as reported in a recent Biotechniques paper (Hernandez et al., 2013). At the moment I started testing my first primer's pair that apparently work well using as template a bisulfite treated DNA (whose methylation status is unknown). To be sure that primers recognize with the same efficiency methylated and non-methylated alleles before starting with my patients' DNA I ordered the Human Methylated & Non-methylated DNA Set from zymo research. I treated the fully methylated, the non-methylated DNA and a combination of both (50:50) using the EZ DNA Methylation-Gold™ Kit again from zymo and try to PCR amplify them.

The result was in my opinion very strange. While the fully methylated DNA amplify well with my primers I can not say the same for the non-mehtylated and for the 50:50 combination. The BS treated non-methylated DNA revealed a very faint band while no product at all was obtained for 50:50 combination. I was worried that something happen in the BS conversion and/or purification so I repeated the treatment but I got the same results in PCR.



Then I tried to use the DAPK1 primers included in the Human Methylated & Non-methylated DNA Set and apparently they work fine (I'm a bit in overcycling, but it doesn't look as differences were present among samples).

At the moment I don't know ho to proceed. Should this data indicate me that my primer combination is more prone in amplifying methylated alleles compared to the non-methylated one? Is it conceivable that the two alleles show such a difference? Do you have any suggestion on how to proceed?



Thanks in advance for any help
eyes
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Re: Different efficiencies for fully methylated and unmethyl

Postby VikR » Mar 17 2017 7:54 pm

Have you considered the following when designing your primers?

Bisulfite conversion = loss of complementarity


Original Template Converted template

5’-GGATCCCGGAATTC-3’ Bisulfite conversion -> 5’-GGATUUUGGAATTU-3’
3’-CCTAGGGCCTTAAG-5’ 3’-UUTAGGGUUTTAAG-5’
Mismatches xx xx x xx x

YOu will need different primers for PCRing the bisulphite converted DNA
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Re: Different efficiencies for fully methylated and unmethyl

Postby r.rosati » Mar 18 2017 12:11 pm

Hello!
Apologies if I didn't get your post right. Having primers that match a final CpG is ok if you want to create methylation-specific primers, but if I understood well, you're aiming at having methylation-independent primers, is this correct? In this case, Hernandez's paper suggests (emphasis mine):
"Methylation independent PCR (MIP) primers should be designed to allow the amplification of bisulfite-converted DNA regardless of methylation status. Primers should also not bind regions containing CpG dinucleotides (Figure 2A) (25) and should flank a sequence of converted DNA containing as many thymines originating from the conversion of non-CpG cytosines as possible (25)."


So if your primers end with a CpG, it is somehow expected that they'll work with fully methylated DNA, where the C would resist deamination to uracil; but not with fully unmethylated DNA, where bisulfite would convert the C to U.
Regarding the 50:50 mix, it "should" amplify from the 50% methylated DNA. I would suspect some contaminant carry-over but you mention that a control primer pair is working fine, so that's not the case. If you get a smear, it might be that some spurious products are interfering with the PCR; but it's hard to say.
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Re: Different efficiencies for fully methylated and unmethyl

Postby eyes » Mar 18 2017 2:05 pm

Hi r. rosati. Thanks for your reply. You understood right since I aim at having methylation-independent primers. Your comment is therefore absolutely correct. However I just not reported in my first post that the primer that included a CpG within it's 5' is a degenerated one so I ordered a primer with a Y in the C position of the CpG nucleotide. This was the reason why I though the results was in some way strange
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Re: Different efficiencies for fully methylated and unmethyl

Postby r.rosati » Mar 18 2017 2:27 pm

I see. Would there be any chance that, while the primers with a C in that position anneal well to their target, the primers with a T get some nonspecific amplification that hampers production of their target?
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Re: Different efficiencies for fully methylated and unmethyl

Postby eyes » Mar 18 2017 3:45 pm

Yes it could be of course. I 'm wondering if should I give up with these primers or I should work further with them maybe changing the enzyme for amplification (I'm using Amplitaq Gold currently).
It would be interesting to set up the system directly in qPCR that should allow to see the amplification of fully methylated and unmethylated alleles in the 50:50 mix by melting curve analysis. Do you have some experience with such an approach?
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Re: Different efficiencies for fully methylated and unmethyl

Postby r.rosati » Mar 20 2017 4:49 pm

Hello,
I'm afraid I don't. But I would imagine that your region of choice contains many CpG dinucleotides, and thus you could spot various degrees of methylation by high-resolution melting. It seems like an interesting idea.
About the primers, I would personally try another pair if possible.
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