Allele specific cloning efficiency after bisulfite treatment - (Sep/20/2005 )

Hi,


I hope my question is appropriate for this bioforum. I performed a bisulfite treatment on genomic DNA (EZ methylation kit, Zymo) and performed nested-PCR on that genomic DNA to amplify specific imprinted genes.

I sent the PCR product to sequence and I could see on the Chroma file that some T/C peaks were juxtaposing each other...involving that I amplified both alleles in the PCR reaction (as was previously discussed here).

I cloned the PCR product in P-Drive and sent 10 clones to sequence...and the results seems weird to me...far from the 50/50 I was expecting... I have to mention that I had a lot of trouble getting 10 clones to sequence...bacteria would not grow easily and the yield of my minipreps were very low (for some samples I had to do 20 minipeps to get 10 to sequence...but miniprep issuesare not the point here...)

The point is: Is it possible that cloning efficiency depends on the allele inserted in the cloning vector? Can some "cloning bias" be introduced such that one bisulfite-treated allele is more easily cloned than the other?

Anyone has an idea about that? Or got stuff that looks like mine??

Thanks!

I am curious what differences are there between the two alleles, different methylation, polymorphism?

When we deal with imprinted genes, we may expect different methylation patterns between the two alleles depending on the maternal/paternal origin of the allele...one allele may be hypermethylated and the other hypomethylated.

Chroma files showed that I'm amplifying both alleles in my PCR... (if you refer to my first posted message)

So is it possible that cloning efficiency is not the same for both alleles after bisulfite treatment?

Hi Drdo,

I don't know if there is a clone bias for methylated allele. It may be possible. But, the under-representation of unmethylated allele in your sequencing clones may due to sampling error because you hardly got 10 colonies in total. I usually don't have any problem obtaining more than hundreds of colonies for sampling. You may want to try Topo cloning kit. I found that result from clone sequencing is consistent to that from direct sequencing.

Yeah I thought it might be some cloning issues...but I also thought it would not harm to investigate about other possibilities.... I'll look for that Topo cloning...thanks pcrman!