Hi Roy,

You need to think about the type of sample, and the type of reaction you use.

Usually during DNA extraction you perform a proteinase step that will destroy your nucleo-protein complexes and therefore your histone marks will be lost. To analyse histone marks you will need to follow specific protocols such as ChIP (chromatin immunoprecipitation) which allowed you to isolate fragment of DNA associated with specific histone marks. You will then remove the histones to amplify and analyse the DNA. therefore during your PCR steps you are already using histone deprived DNA for input.

For DNA methylation, you perform a PCR in a context deprived of the machinery recognizing and reproducing the DNA methylation profile. Therefore PCR will not conserved your methylation. That is the reason why, we often use whole genome amplified DNA as unmethylated control in experiments. Because methylation is not conserved during PCR, you need some trick to detect it ... either Bisulfite conversion (which deaminate all unmethylated cytosin into uracyle... creating a differential template between unmethylated and methylated sequence, and therefore recognizable by PCR), or using restriction enzyme which have specificity toward methylated or unmethylated template. There are other methods available using other tricks, but these two approaches are the most common.

I hope it answer your question,

Best,

Ludovic

Dear Roy,

When you perform PCR reaction you recreate the replication process in vitro using only the necessary substrates (usually DNA polymerase, dNTPs, DNA template, starters). The composition of this reaction excludes the possibility of maintaining DNA methylation due to lack of DNMT1 (DNA methyltransferase I), which participates in postreplicative maintenance of DNA methylation. The same applies for maintaining histone modification patter in PCR. The absence of HDAC (histone deacetylase), HAT (histone acetyltransferase), HMT (histone methyltransferase) and other histone modifying enzymes does not allow for the recreation of maternal histone modification patter. However, I do not exclude the possibility that it is/will be possible to maintain DNA methylation or histone modification pattern in some modification of PCR.

Kindly,

Paulina

PCR reactions use naked DNA, so there are no histones present in the template and no histones in the reaction mix.  As stated above, DNA methylation is also not replicated by PCR. 

BTW, the same is true for  other DNA modifications like 5-hmC (hydroxymethyl), 5-fmC (formylcytosine) and others: they are lost during PCR.

Hi Roy,

When you extract your DNA, you deplete it from the all the proteins that bound to it. therefore all your histones are lost during this process. likewise, when you perform PCR, you loose your DNA methylation marks because the cytosines in you dNTP mix that you add to your PCR mix are not methylated, so you replace mC by nonmC during amplification.

If you wanted to measure DNA methylation within a fixed location of the genome, Methylated DNA Immunoprecipitation (MeDIP) is a useful technique. Otherwise whole-genome or targeted bisulfite sequencing is the way to go. 

epiGBS?

It is related to DNA methylation.