I want to check the global methylation status in some cancer cell line. I want to know whether I can use direct sequencing of PCR product of repetitive elements after Bisulphite treatment for global methylation or have to do pyrosequencing? I want to know why pyrosequencing is preferred over direct sequencing? and also what are the disadvantages of direct sequencing? what is the difference between direct sequencing of PCR product and firstly cloning and then sequencing the PCR product in case when we do not want allele specific methylation as direct cloning gives an average of methylation while cloning method gives methylation status of each DNA molecule.So among direct sequencing, cloninig and then sequencing and pyrosequencing, which will be the best choice and why?
if you want go for global methylation, then till date pyrosequecing is the best method. By simple sequencing, there are chances that you will miss a methylated CpG island or a single base but pyrosequencing is very sensitive.
Hi,
- The first thing to keep in mind is that direct sequencing is not quantifying. You will have a qualitative output but no possible quantification. There is possible post-quantification of the electropherogram which was used in the past (Lewin et al.), but I am personally sceptical of the reliabillity of such post-quantification and I think it should be validated for any locus using a methylated/unmethylated scale.
- The second point is that direct sequencing (without cloning) usually results in poor output (dirty traces). It is then recommended to do the cloning which also allow you to have a quantitative aspect (ref Frommer et al.).
- Third, if you want to use the repetitive element as surrogate for global methylation, you have to consider that the amplification step is not trivial. Your primers are annealing to the most conserved part of your repetitive element so you screen as much of the genome as possible. You will get a lot of noise because conservation is not complete.
Therefore optimization is the key.
Pyrosequencing has already been optimized for it (ref Tabish et al.), and is commonly used. Then to get easily your results, and for reproducibility and discussion purpose, I would suggest you to go with this.
I hope it helps,
Best,
Ludovic
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Hi Lalita,
I discourage you from direct sequencing of PCR products containing repetitive elements after the bisulfite treatment. This is because:
- repeats are both genetically and epigenetically polymorphic.
- mutations occurring in different copies will yield double peaks an ambiguos Sanger spectra.
- Methylation levels may differ between individual copies. This will result in complicated interpretations.
One possibility is to design primers annealing to unique regions flanking a repeat. In this way, perhaps, direct sequencing is feasible. If your primers anneal to repetitive regions, cloning and sequencing is probably inevitable.
Examples of bisulfite analysis of repetitive ribosomal RNA genes are in papers attached
Best
Ales
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