Science Buddies: "Ask an Expert"

Hello! I Would like to know if someone can explain how i could verify if a specific gene like hexokinase 2(HK2) is silenced or not in terms of regulation by methylation and acetylation? I probably would have to design a specific primer to this gene but i don't know how i would identify the promoter region of this gene. Also it would be important to identify CpG islands near the operon and which histones are involved in repression. My question is more related with techniques that i would need to use if I did a study in this area that is new for me. I have the idea that I can use a variant of PCR but i'm not sure about the process.
The main propose is to identify the influence of epigenetic in a specific gene like HK2 in a case of cancer.

Thanks in advance.
DFAS

The hexokinase promoter sequence is likely known, so we can design PCR primers specific for it if required.

Promoter methylation can be detected via several methods: http://www.neb.com/applications/epigene ... ethylation

The best is bisulfite sequencing: http://en.wikipedia.org/wiki/Bisulfite_sequencing

There are other methods for which you can purchase kits, that have some advantages in terms of time and costs but which will give you less precise information:

eg http://www.thermofisher.com/order/catalog/product/K1441 (size of bands cut with the restriction enzymes will tell you if DNA methylation differs between two samples).

and http://www.thermofisher.com/order/catal ... ct/ME10025 (MBD2 protein binding enriches methylated DNA, which can then be amplified by PCR to look for specific regions.

There are also a number of commercial kits available for assaying histone acetylation as well.

Let me know if this helps and how this affects your plans.

Cheers,
Colin

Thanks Colin I think that I was able to design the project.

Once more thanks for taking time to answer it.



Cheers
DFAS