Starting with DNA methylation analysis - (Jun/15/2005 )
Hi,
I'm a new memeber of this nice discussion forum. I have some problems that I will be very happy if someone can help me to solve....
I am trying to start with DNA methylation analysis and it is not working 
I am using 1 µg DNA that is extracted from blood cells and I am using the EZ DNA methylation Kit for bisulfit treatment. I have tried both the Bisearch and Methprimer for designing primers. The lenght of the primers are 23 and 24 bp. I have tried to dilute the DNA before the PCR (10 times) and also not to dilute it. I have used AmpliTaq and Gold Buffer from Applied. But after the PCR I don't get any bands when I do an electrophoresis. So my questions are:
Q1: Is there a way to measure the bisulfit treated DNA, before doing a PCR?
Q2: After the PCR´, is it possible to se bands on an agaros gel?
Q3: Doing an optimization is it in the same way as with ordinary DNA?
Hope someone can help me! Thanks!
Hi Saga,
I have a few questions for you.
1. are you performing MSP or bisulfite seqeuncing?
2. Can you post your sequence and primers?
3. Did the kit include a positive bisulfite control and primers and were these run beside your samples? you should check if your PCR is working.
4. are you performing one round or two rounds of PCR?
Some answers:
I am not familiar with the kit you are using, if it claims to have no loss in DNA quantity then you could spec it. Otherwise a more sensitive assay is to use the Picogreen quantitation assay.
Good luck!
Nick
Hi Nick,
Thank you for your answer!
I am doing bisulfite sequencing.
I have no positive control. How can I use a positive control? I'm a total beginner in this area....
I'm only performing one round with PCR, 40 cycles.
I measured the DNA after the bisulfite treatment with the picogreen assay and according to that method I had DNA in the samples. But what makes me confused is that the picogreen assay should only measure dsDNA and after the bisulfite treatment the DNA is ssDNA. Whats your opinion about this?
Cheers!
I have a few questions for you.
1. are you performing MSP or bisulfite seqeuncing?
2. Can you post your sequence and primers?
3. Did the kit include a positive bisulfite control and primers and were these run beside your samples? you should check if your PCR is working.
4. are you performing one round or two rounds of PCR?
Some answers:
I am not familiar with the kit you are using, if it claims to have no loss in DNA quantity then you could spec it. Otherwise a more sensitive assay is to use the Picogreen quantitation assay.
Good luck!
Nick
Hi SAGA
Q1: Is there a way to measure the bisulfit treated DNA, before doing a PCR?
A: You don't need to. I would say if you do, the amount of DNA is too little to be measured reliably. I've never used the EZ kit but at one time I was about to switch from Chemicon's kit to EZ kit. I didn't after reading the EZ kit's instruction which says "elute DNA in 10 ul (if I remember correctly) solution". Come on, 10 ul is only for 5 PCRs. You may ask the company to see if you can dilute the modified DNA to make it last longer.
Q2: After the PCR´, is it possible to se bands on an agaros gel?
A: You said you ran one round of 40 cycles. You may not be able to see a band after 40 cycles. You can either add Taq after 40 cycles and run another 5-10 cycles or do another round of PCR (25-35 cycles) using the first PCR as a template.
Q3: Doing an optimization is it in the same way as with ordinary DNA?
A: Should be.
Good luck!
Hi Saga,
I am not familiar with the EZ kit however I have used MethylEasy and it comes with positive controls (ie: bisulfite treated DNA and primers for a PCR). I am surprised the EZ doesn't come with one!
It is very difficult to create your own positive control, do you know of anyone who has performed bisulfite PCR routinely and can spare you a little DNA and primer? (this is just to test if the PCR mix is working.)
What is the Tm you set for the PCR reaction itself? I normally set 2C lower than calculated, and I have gone down to 10C lower to get a product.
I don't use methprimer for BSP because I find the primers it selects are very lowsy.
Bisulfite requires denaturation of DNA for it to react with the cytosines, by the end of the protocol, the DNA will have renatured albeit severely mismatched. Besides, Picogreen is able to detect single stranded nucleic acids as well (there is a specific kit for RNA though i think they are all the same)
If you could post your sequence and primers you are using for the PCR it will help alot to find out what's going wrong.
Nick
Hi,
I get the PCR to work 
I changed from AmpliTaq gold from Applied to Qiagens HotStar! I could see very clear bands on the gel after just one round with PCR. I was expecting the primers to be very unspecific and that I should have a lots of bands on the gel. But I only had distinct bands. I was expecting lots of optimization rounds....Someone that has comments?
Now I am continuing with sequencing. And I really hope it will work!
I have read that there are almost only C in the combination CpG that are methylated, but that C not combined with G also can be methylated. My question is how common it is with methylated c (not combined with G)?
I am using the methprimer for primerdesign. )I tried the bisearch but I like the methprimer more. Nick why don't you like the methprimer?
PCRman, it is true about the EZ methylation Kit. I eluate the DNA in 10 µL and I agree its very tiny. But I have dilute the DNA 10 times with water and it works. I have also heard that you can dilute it even 20 times.
Thank you!
/Saga
I am not familiar with the EZ kit however I have used MethylEasy and it comes with positive controls (ie: bisulfite treated DNA and primers for a PCR). I am surprised the EZ doesn't come with one!
It is very difficult to create your own positive control, do you know of anyone who has performed bisulfite PCR routinely and can spare you a little DNA and primer? (this is just to test if the PCR mix is working.)
What is the Tm you set for the PCR reaction itself? I normally set 2C lower than calculated, and I have gone down to 10C lower to get a product.
I don't use methprimer for BSP because I find the primers it selects are very lowsy.
Bisulfite requires denaturation of DNA for it to react with the cytosines, by the end of the protocol, the DNA will have renatured albeit severely mismatched. Besides, Picogreen is able to detect single stranded nucleic acids as well (there is a specific kit for RNA though i think they are all the same)
If you could post your sequence and primers you are using for the PCR it will help alot to find out what's going wrong.
Nick
Hi Saga,
with non CpG methylation you would normally observe this in plants as it is more common and it is usually at CpNpG seqeunces. In mammals, there are a couple of papers describing such a phenomenon, assymetric methylation, I believe it to be an artefact of the assay.
I don't really believe in methprimer's primer selection because when I tried a couple of sets and seqeunce amplicons from them, I got mixed results (ie: lots of non-CpG methylation suposedly and this was what I was not expecting)
Remember, the bisulfite reaction is not 100% efficent and if your primers bind to unconverted template preferentially then the seqeunce results you get will be ambiguous. Primers should be designed to favour templates that are fully converted and this is not achieved by methprimer I believe.
Good luck
nick
Congratulations, SAGA!
Thank you for let me know that you can dilute the modified DNA by EZ kit. In that case, the amount of recovered DNA is not a problem with the kit and I will give it a try. Then it should be simpler than Chemicon kit.
To Nick:
Designing primers on bisulfite modified DNA is really a tough job. Any program faces the same limited choices. Sometimes you have to sacrifice some criteria (which are important in terms of regular PCR) to just allow the program to return some primers to you no matter how good they are. Regarding methprimer's ability to differentiate modified vs unmodified DNA, it provides a parameter "primer non CpG 'C'" with a default setting of 4. Certainly you can increase it if you need high stringency.
pcrman,
totally agree about your point on programs being able to pick suitable primers. I haven't used methprimer for a long time as I have been picking primers by eye. I was not aware about the non-CpG C feature, I'll go back and have a play with that and see how things go.
Cheers!
Nick
How do you design primers just by your eyes?? any special criteria?
i tried to use primer 3, but it always said "INPUT PROBLEM: Too many elements for tag EXCLUDED_REGION". i dont know how to solve the problem.
on the other hand, i found its a bit hard to use peal primer, not very user-friendly i think.