What is the max. amplicon size for CpG sites after bisulfite treatment?
I have to sequence them after amplifying them through PCR. But I have "sanger" sequencer.. not pyrosequencer which complicates the situation.
These are the CpG sites I will work with (cg02228185 in
ASPA, cg25809905 in ITGA2B, and cg17861230 in
PDE4C)
I also want to know what PCR is the best method for this whole experiment.
Just standard PCR, qPCR or something else?
Your answer will be very appreciated.
Max amplicon size will depend on the size distribution of the DNA fragments obtained after bisulfite conversion. This will depend on the typical size of the input fragments and the "harshness" of the bisulfite conversion procedure. In my experience, the Zymo EZ-DNA methylation Gold kit has worked well on high molecular size genomic DNA. They also have a "beginners" kit that includes control samples and primers:
http://www.zymoresearch.com/epigenetics/dna-methylation/bisulfite-conversion
As told before max. amplicon size is limited by bisulfite rxn. Anyway it also depends on the technology one will use downstream to analyze the fragment! A basic rule for Pyrosequencing and MassArray could be: the shorter the better! Methylation analysis needs experience - so contact a group or company is doing this business a long time to get reliable data! These guys also know about hemi-methylated DNA and other findings ...!
Hi ChanWoo,
I agree with previous comments, and I will add some others:
- For the size, as Uwe mentioned, usually the shorter the better... Why? because after bisulfite conversion you will have a massive decrease in complexity of the sequence from 4 base code to mostly 3 base code. therefore mispriming of your oligos are likely to occur and the longer the sequence the higher the probability to find recurrent motifs.
- You also need to consider that direct regular sanger post bisulfite give usually very bad results. For this reason people usually clone their fragment which also allow to have a quantitative results since you sequence individual molecules. However you can easily imagine that adding this step is a lot more cumbersome.
- However from what I see of the CG site you want to look for, two of them are what I would call the lonely lolipop (meaning their are in the sea, not in a CpG island). Therefore you might want to direct your self towards a more simple method which is not really used anymore but worked fine, which is the Ms-SNuPE (publication attached). If I recall correctly you can also use it on your capillary electropherosis machine.
I hope it helps,
Best,
Ludovic
Article Methylation-sensitive single-nucleotide primer extension (Ms...
We described a single multi-purpose PCR-based integrity assay both on native DNA and bisulfite treated DNA (doi: https://doi.org/10.1101/168195). By performing the first assay on your non treated DNA and the second on your bisulfite treated DNA you will get some info about the amplificabillity your DNA before and after the treatment. Based on the second assay you can decide on the length of your amplicons for bisulfite sequencing. Don't hesitate to contact me for feedback.
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