Hi Alexandra

maybe you're right to say that only a low proportion of your samples could be methylated. In this case the results will be in the range of your background, impossible to detect it in sanger sequencing. one way would be to clone, but cloning what? you won't be able to have an idea on the percent of methylated cells. the best way would be to analyse your samples in chip-seq experiments, the coverage would give you the percents of cells harboring the methylation, if it is.

fred

The direct bisulfite sanger sequencing technique relies on the peak height of methylated cytosines and unmethylated cytosine which upon the bisulfite treatment convert to Thymines.

So you have to use this equation for each CpG dinucleotide to determine the methylation rate for each CpG dinucleotide:

Methylation rate= the cytosine peak height (Forward primer) or guanine peak height (Reverse primer), divided by the peak height sum of cytosine and thymine (Forward primer) or guanine and adenine (Reverse primer). (kacevska et al. 2012).

The propose of cloning in this technique is to cover up the limitation of sanger sequencing which can not sequence the beginning and the end of the desired amplicons. Hence by cloning the desired amplicon sequencing start from a few nucleotide before and end in a few nucleotide after the amplicon.

Also, you have to keep in mind that detection limit of this technique is about 10 percent which means that it's not possible to evaluate the methylation rate below than 10 percent.

the final word, yes,  you are right about your cells. currently, you are examining every nucleated white blood cells. In my opinion, you have to select one of them for further analysis and separate them by techniques like flow cytometry or something like that.

Good luck

Hi Alexandra,

As I previously mentioned in other question of this type, direct bisulfite sequencing can be useful if you want an idea about how the methylation looks like at a loci, however you should forget about it if you want reliable quantitative results.

The first reason is sanger IS NOT quantitative. This is true for diverse reason, bias in amplification according to template (which will be bigger when you talk about bisulfite treated template), bias in intensity during the sequencing (look at your raw sequencing data and you will see a decrease in intensity at the 3' of your amplicon - you synthesize more easily the short fragment than the long ones). and finally the direct sequencing after bisulfite usually gives relatively dirty results because of unspecic annealing of your sequencing primers (due to loss of complexity after bisulfite treatment).

As suggested previously, people tried to compare peak intensity to obtain % of alteration but this a rough estimate and the 10 % sensitivity mentioned earlier is I think very generous... you should consider closer to 20-25%...(but that is my personal opinion - experience).

You need to ask yourself what do you want to see and in what...

From what I read in your question it seems you're interested in environmental exposure and consequence on the methylation... so epigenetic epidemiology... Just look at paper in this field you will see that the variation observed in this type of studies are usually very small 3-5%. Therefore you direct sequencing will not be good enough. And if you go through cloning just consider that you will need to clone around 100 colonies per samples...

As you mentioned you also need to take into account that whole blood DNA is a mixture of cells, therefore you just have an average signal of all these cells. If you look at the papers of A. Houseman for example, you will realize that the different blood cells do not have the same epigenetic profile therefore it can affect your results.

If you want to see whether you have methylation at this loci (if you doubt about it), run a MSP... it will not be quantitative but it will be sensitive, easy and fast... if no amplification is visible in with the Methylated primers, you'll have your information about the methylation status.

If you get an amplification and you want to understand how much, you have a lot of different quantitative techniques with different sensitivity... Several years ago I wrote a book chapter with my colleague about it... It is not updated with the most recent techniques but it might help you.

I hope it helps,

Good luck

Chapter Laboratory Methods in Epigenetic Epidemiology

Please see http://www.biotechniques.com/BiotechniquesJournal/2013/October/Optimizing-methodologies-for-PCR-based-DNA-methylation-analysis/biotechniques-347156.html