About MSP positive control - Choose what to methylate as positive control (Jun/27/2005 )
I am doing methylation research about cervical epithelium by MSP.
Now I need a positive control for the MSP.I perpose to purchase Sss I (NEB) to conduct positive control. But I wonder what sample can I use Sss I to methylate ??? Do you have good suggestion?
ideally the promoter or gene you are looking at with your msp primers, are you able to amplify and clone this into a vector which you could then SssI methylate?
Thank you very much Nick!
But I still have questions. Amplify the promoter and clone this into a vector, is it the best way to constitute the MSP positive control? Is there any simpler way for the positive control? AND if I use the vector to constitue the positive control,which company's vector should purchase?
With MSP you need to know your primers work properly so if you were to choose a different region and SssI methylate this you will require a new set of MSP primers. This is not entirely a true positive control.
What I am proposing is that you amplify your region of interest with normal PCR primers that include your region and the sites of you MSP primers. Clone this into a vector (any will do) like pGEMT, isolate the vector with the insert, SssI methylate this, then bisulfite treat this along with your test samples and run an MS-PCR on both samples, this is a true posistive control as you are able to tell if your MSP primers are in fact MSP!
Apart from this I don't think there would be an easier way!
I followed your suggestion to constitute a vector with target region which I am interest in,and then methylated with SssI .But the result is not good. so can you send the protocol that SssI methylate the vector to me?
or you can send a few reference papers to my mail box . email@example.com
thank you very much!
the protocol I used is included with SssI methylase. Essentially, I take 2ug of isolated plasmid DNA and methylate using SssI according to the included protocol which is (to quote from NEB):
Reaction & Storage Conditions
1X NEBuffer 2
Supplemented with 160 ?M S-adenosylmethionine (SAM)
Incubate at 37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM dithiothreitol
pH 7.9 @ 25°C
One unit is defined as the amount of enzyme required to protect 1 ?g of ? DNA in a total reaction volume of 20 ?l in 1 hour at 37°C against cleavage by BstU I restriction endonuclease.
A good way to see if the DNA is indeed methylated is to perform HpaII/MspI digestion, the plasmid will fragment with MspI but not with HpaII.
As for papers, I don't have any right now at hand that has published any work silmilar to this except by NEB describing the enzyme!