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[Spliffeh]

Hy everyone,
I am entirely new to the entire methylation checking business so apologies if my use of words isn't correct.

I have to study the methylation status of the following gene,
Gene

I have read quite a few topics here on this forum but it still isn't entirely clear to me how I should start on this.
For example, I have no knowledge of any methylation sites on this gene so I assume it's best to go for BSP and not MSP?
The goal is to do a DNA sequencing of a control and a treated sample.
Is it necessary to do a nested PCR?
Let me know if you need more details,
thanks in advance!

-Spliffeh-

[k_undertoe]

Spliffeh, on Feb 1 2010, 08:29 AM, said:

For example, I have no knowledge of any methylation sites on this gene so I assume it's best to go for BSP and not MSP?

The goal is to do a DNA sequencing of a control and a treated sample.
Is it necessary to do a nested PCR?
Let me know if you need more details,
thanks in advance!

-Spliffeh-

Yes, I would definitely use BSP- MSP is artifact prone if your primers are not absolutely stringent and accurate, very apt to mis-prime IMHO. For BSP, you do need to do 2 rounds of PCR to get your target, so yes it will need to be nested. Check the tool MethPrimer as a starting place to design your primers.

Also, a look at this thread here might be useful.

Cheers!