All scientific experiments need controls. Specific controls are required for certain type of experiments, such as DNA methylation assays. DNA methylation is a powerful, well-studied epigenetic mark that continues to provide extensive insight into transcription regulation, embryonic development, genome stability, and chromatin structure. Changes in DNA methylation profiles have also been linked to the development of many diseases 1. Many clinical applications have utilized DNA methylation as a biomarker for early detection, diagnostics, and personal therapy 2, 3. This requires accurate characterization of methylation patterns at a specific region or single-base level.
If you want to learn more, please visit our website TSKT.
Most commonly used DNA methylation assays include bisulfite sequencing using NGS technologies, methylation-specific PCR (MSP), and methylation-sensitive restriction enzyme (MSRE) assays. To minimize bias in quantifying methylation levels, especially in clinical applications, each assay needs to be carefully designed and optimized to ensure its robustness4. Integrating established controls is key for optimizing assays and monitoring assay’s effectiveness . For optimization of DNA methylation assays, it is best to use established DNA standards with known methylation levels. Typically, this requires a methylated and non-methylated DNA standard, which contain nearly 100% and 0% methylation, respectively, at all CpG sites. The use of these two standards individually or in combination can help identify potential biases in assay development. For established workflows, these standards can serve as a positive and negative control to validate the procedures and results, which is essential in clinical testing to enhance quality control and assurance.
As a good practice, methylated and non-methylated standards should be processed in parallel to experimental samples as a quality control for the whole workflow. In contrast to standards that are expected to produce a consistent result each time, experimental samples can have many variables, making troubleshooting difficult. In PCR-based and NGS-based DNA methylation assays, inhibitors carried over from the samples, primer design, enzymatic reagents, instrument failures, and many other factors can cause an assay to fail. Data produced by the standards can suggest where to start troubleshooting. For example, if standards perform as expected when run in parallel with experimental samples which perform poorly, the workflow is functioning properly, suggesting the experimental sample quality may be an issue. If standards did not perform as expected, there may be an issue within the workflow that caused the assay to fail.
Bisulfite PCR (BSP) is a common methodology used for methylation analysis at single-base resolution. Samples are first bisulfite converted and amplified using bisulfite-specific primers. The PCR products generated are sequenced (i.e. Next-Gen Sequencing, Sanger Sequencing, pyrosequencing) and analyzed to determine the methylation status at every cytosine. However, it is critical that primer pairs designed for BSP amplify both methylated and non-methylated sequences with the same efficiency (Figure 1). Otherwise, any amplification bias can result in skewed methylation levels.
Using methylated and non-methylated DNA controls, BSP primers can be easily optimized (validated) based on following criteria:
Unlike bisulfite PCR, methylation-specific PCR (MSP) requires methylated and non-methylated templates to be differentially amplified by two different primer sets. One is a methylation-specific primer set, and if amplification occurs, it indicates the amplified region is completely methylated. The second primer set targets non-methylated templates, so any amplification with this primer set indicates the amplified region is completely non-methylated. If amplicons are generated by both primer sets, the region is partially methylated.
Such two primer sets required for MSP can also be optimized and validated utilizing Methylated & Non-Methylated DNA Controls using following guidelines:
During primer validation, having characterized methylated and non-methylated DNA standards can streamline the process. For example, if a slight PCR signal is observed using the methylated standard with the non-methylated primers, or vice versa, it would indicate that the primer set is non-specific. For established workflows, especially in clinical assays, methylated and non-methylated DNA standards can serve as positive and negative controls to validate the MSP workflow. Standards will help confirm that the entire process – bisulfite conversion, amplification, and analysis – are functioning appropriately, and the results for the test samples are reliable (Figure 2).
In addition to validating primers, DNA standards can be used in place of precious samples to optimize the annealing temperature for new primer sets (Figure 3). An annealing temperature gradient should be performed for each newly designed primer set to ensure optimal amplification of the intended target.
For more tips on primer design, see the Bisulfite Beginner Guide.
Methylation sensitive restriction enzyme (MSRE) assays rely on restriction enzymes being able to distinguish methylated versus non-methylated cytosines. If the restriction site is not methylated, the enzyme will digest the DNA while methylated sites will be uncut. Then, methylation at the site can be evaluated using quantitative PCR by comparing the amplification of the digested versus undigested sample. The smaller the ∆C(t) between the two, the higher the methylation levels are, and vice versa (Figure 4). DNA visualization methods, such as an agarose gel, can also be used to determine if the digestion occurred at the region of interest.
Methylated and non-methylated DNA standards should be digested in parallel to experimental samples to demonstrate the robustness of the restriction enzyme in distinguishing methylated versus non-methylated sites. This will also help determine the sensitivity of the assay to the region of interest.
DNA standards can be used for optimizing a variety of other applications as it is high-quality genomic DNA with known methylation patterns. This makes them ideal substitutes for precious DNA or problematic sample types when titrating experimental conditions. For example, when optimizing bisulfite library preparation of FFPE DNA, the DNA standards can easily be sonicated or fragmented to be representative of the experimental samples.
For more information, please visit DNA Methylation Kits.
The methylated and non-methylated DNA standards have been adapted as controls for numerous methylation assays:
The Human Methylated & Non-methylated DNA Standards are an example of a validated control set for methylation analysis of human samples. They have been widely used for all the assays mentioned above and much more. Zymo Research also provides a range of other DNA standards that can be used for optimization or quality controls for other assays to help guarantee your data is of the highest quality and ready for publication.
Still unsure of what standards work best for your application? Please Technical Support ( ) for help!
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!
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
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.
If you want to learn more, please visit our website Immunology Kits.
-Samantha-