Hello everone,
actually i want to do analysis of epigenetic alterations and i dont knw exactly which kind of sample i should take cz people are using serum,biopsies.but biopsies r not easy to get so wat shoud i do.
2ndly for bisulfite seq,can i use the bisulfit converted products amplicons and use the same sequencer as we use for dna n rna sequencing not any special sequncer and does it require any insertion of any special chemical during sequencing?
what taq shoud be used for pcr?
how methylted n unmethylated primers actually work?i have seen a video of abnova but it doesnt explan that methylated and unmethylated cpgs can be on the same strands
-madi_epi-
you should be able to sequence as normally performed (at least with sanger sequencing).
-mdfenko-
Yeah well you have to take the bisulphite conversion into account when youre designing your PCR and sequencing primers. Because al cytosine outside CpG context will be converted into thymine, and these will also serve as a control for completion of conversion. If your primers contain multiple converted cytosines the PCR will only work on converted DNA.
Also try to avoid CpG sites in your primer sequences because you dont know the methylation status of these sites (I assume).
I did have some trouble sometimes with sequencing bisulphite converted amplicons since the sequence contains a very high amount of A/T basepairs and the sequencer has had some trouble with that.
Good luck :)
-Forensic_Fleur-
1. You should compare serum and biopsy. Then determine which sample is more relevant for your situation.
2. Determine the sequence you want to target. Design primers using MethPrimer -
http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi. Input DNA, the software will bisulite convert in silico and design the primers. Try to keep the amplicon size around 400-600bp. Bisulfite convert your DNA with a kit or make the reagents. PCR amplify your bisulfite converted DNA with primers designed by MethPrimer. Run the product on the gel and gel purify the band. Clone this into a vector (i.e. TA Cloning). Picking 10 individual colonies (i.e. 1 clone would be equivalent to 1 allele) and sequence the insert from the isolated plasmid (traditional sanger sequencing). Your sequencing core may have software to help with the peak calling. You can then compare the 10 sequences from one sample using CpGViewer - http://dna.leeds.ac.uk/cpgviewer/, to get an overview of the methylation status. 10 sequences per sample would allow you to determine the methylation status of 10 alleles.
3. The taq does not matter. If you are going to perform TA cloning then a non proofreading taq is needed to add the A overhangs. Otherwise standard taq works fine. Hot start may help but is not necessary.
Methylated and unmethylated primers are for methylation specific PCR. F primer overlaps a CpG site at 3' end. Two F primers would be designed - methylated will have a CG and unmethylated a TG. You would run two PCRS, one with methylated primer and one with unmethylated primer. If CpG is methylated then only the methylated primer would anneal and produce a PCR product, while he unmethylated primer would not anneal and no product. Only allows you to interrogate a single CpG. Bisulfite sequencing is much more information but more work.
Hope that helps!
-5280-
