Primer sets for Bisulfite-modified and unmodified DNA - (Feb/12/2007 )
I'm relatively new to gene methylation studies.
My project centres on monitoring the effects of some (presumably) non-genotoxic carcinogens on primary human hepatocytes alongside the demethylating agent, 5-azadC.
My major genes of interest are p16, p21, Rb, c-myc, K-ras and Erb.
I have used the methprimer programme to design some M & U primers for MSP analyses of bisulfite-treated DNA but I'm just wondering if those primers can be still used on unmodified DNA. Personally, I feel that because the unmethylated C's have all been converted to U's , it will be unwise to use the same primers for DNA that hasn't been modified.
Can I just use primer 3 to design primers for the unmodified DNA (which will be used for comparisn with the bisulfite-modifed DNA after PCR amplification). Or is there any way to use meth primer to generate two separate primer sets, each for bisulfite-modified and unmodifed DNA?
Any comments and advice will be highly appreciated (I'm sorry if this sounds a bit to basic). Kindly help because if the primer design is wrong, then everything else will likely be wrong as well.
MSP primers are designed to recognize converted DNA, thus they will no longer recognize untreated genomic DNA. You use one set of primers for unmethylated and one set for methylated DNA. If neither picks up a signal, then your conversion was not complete.
I second tap14 with his remarks regarding M and U primers for MSP. Primer design is really essentiall for methylation studies and I would suggest that you have a look at ABI Methyl Primer Express (it's free) because it seems to work more appropriate (it takes mispriming with no-modified template into account which Methprimer doesn't).
For the question regarding unconverted DNA - sure you can use primer3. The methylation specific primer design tools normally don't offer this feature. But may I ask, what exactly you need this control for?
Thanks, Krumel and Tap14 for your comments. I will use primer 3 for the primer set for unmodified DNA.
As for your question on why I need to PCR the unmodified controls, here are my thoughts:
After PCR amplification, I need to also carry out bisulfite sequencing analyses on my samples. To calculate the % methylation of test samples, I will need to compare and contrast the sequence data of both the bisulfite-treated and unmodified DNA. In the bisulfite-modofied DNA sequence, all the non-methylated C's would be seen as T's whereas in the unmodified DNA sequence, both methylated and unmethylated C's will both appear as C's only. In this way, one can actually localise the exact sites on the DNA where methylation occured. Does this make sense to you? Otherwise, my thinking may be wrong!
In any case, if you know any better methods of mapping out the regions in a gene where methylation has occurred, then kindly let me know. I believe MSP is for qualitative purposes only while BS sequencing will give more quantitative data.
That's why I have adopted this approach for the time being. In future, I may be looking at real time-MSP, MethyLight etc.
Hope to read your comments soon.
Well, I would suggest that you only perform bisulfite modification and afterwards sequencing (BSP). With MSP you can only analyse the known CpG's within your primer sequence. When choosing BSP, you can check the methylation (quantitative and qualitative) of every CpG within your sequence of interest. You do not need to analyze unmodified DNA (only if you think that carcinogenesis might have altered the sequence), as methylation in mammals can only occur in CpG dinucleotides. When performing direct cycle sequencing, you get an amount of methylation for every CpG-C while you can check for appropriate conversion by looking at the non CpG-C's that should have all been converted to T. As you work on human cells, the sequence should be known and therefor you can answer the question where a C should be converted (non CpG-C) or may be methylated (CpG-C) in silicio.
MSP only makes sense if you already know, that there is one CpG (or at max 2-3), which methyaltion status is essential for the expression. To check for new genes you have to do BSP. As MSP biases towards methylated or unmethylated sequences, it doesn't make sense to sequence MSP amplicons.
Hope that helps you save time and money...
Once again, I do appreciate your input.
Actually, I was just gonna do MSP to check for the methylation status of my gene of interest.
If it's methylated, then I can do BSP sequencing to analyse the methylation profile. If unmethylated, end of story.
Like you rightly mentioned, I do agree that MSP amplicons are not useful for sequencing purposes.
With the other info you gave about unmodified control DNA, I think I wouldn't really need to amplify them as such.
Surely, some time and money will be saved by our lab.
You can only use MSP as screening method, if you know which CpG's are important! MSP is not really a good screening method... your gene of interest may show heavy methylation but the CpGs you chose for MSP are not methylated and vice versa... Therefor, use BSP to screen.