My overall question is - why are these aldehyde reactive biotinylated probes having high nonspecific interactions with DNA, and how can I remove it prior to my elution steps?
I have been working on enrichment workflows where I bind a biotinylated molecule to AP DNA, which has an aldehyde group used for binding. It appears that there is a very high amount of nonspecific binding of DNA to the beads in the presence of the biotinylated molecule. I react a first molecule with DNA (AP DNA sample and control DNA without AP sites) to add the biotin group for enrichment. Only the first sample should actually bind to it. However, it seems that there is nonspecific binding of control DNA too. I pass the solution through a size exclusion column to remove excess chemical and react it with streptavidin magnetic beads. Then I wash the beads with typical washing conditions in the literature and suggested by manufacturers. The washing buffer is 5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20. Afterwards, I add a second chemical to displace the first one and elute the DNA. My elution conditions are 1 mM of my chemical, 100 mM pH 6.5 buffer at 37C, 850 rpm shaking for 1 hour. However, when I do this, both the DNA samples (AP and control) seem to be eluting based on qPCR data. The reactive DNA is usually 3 Ct values lower than the unreactive DNA, which isn't very high fold enrichment (usually between 10-100 fold). I have repeated this many times and minimize contamination in my qPCR assays (always add an NTC to check), so it isn't an issue of cross contamination between my samples.
To see if it was an issue with the chemicals we use, I reacted the same DNA with an aldehyde reactive probe (ARP, structure shown here https://shop.dojindo.com/products/arpaldehyde-reactive-probe), which is well-documented for this purpose. I repeated the workflow and got similar results - EXCEPT when I incubated the DNA with the same 100 mM pH 6.5 buffer at 37C for 30 mins with 850 rpm shaking. I treated that as a wash step and discarded that sample. These are essentially the conditions I use in the previous assays for elution, just without the reactive chemical. Then, I elute my DNA in 95% formamide at 65C. Using that condition, I then achieved around 12 Ct value difference, giving a very high fold enrichment. This only happens in the presence of the biotin molecules - not just the beads. There is minimal nonspecific binding to only the beads. I haven't seen anything in the literature of people doing a heated wash to decrease the nonspecific interactions, but it seemed to work well for that probe.
I tried that wash then with the chemical I am using, and it seemed to elute both my DNA samples. Increasing the pH to 7.5 in HEPES buffer didn't elute either DNA. Is there any other way to elute the nonspecific DNA without heat or depending on pH? Or is there an optimal pH for reducing nonspecific DNA interactions? Or if someone can just give an idea of why it's binding to the DNA, perhaps I can add some blocking reagent to minimize it.
Maybe the problem is not so much with nonspecific DNA binding but with poor reaction of the AP DNA with the biotinylation probe, so that there is comparatively little biotinylation.
There are a few things you can do to reduce nonspecific binding of DNA to biotinylated beads such as use a blocking agent like bovine serum albumin (BSA), casein, and nonfat milk powder.
Wash the beads thoroughly with a high salt concentration and use higher temperatures.
Thank you for your answers. It ended up still being an issue of pH and heating. The probe I am using it most stable between 7.5 and 8.5 (I was using lower pHs previously), so the heating step was causing it to be removed. Now using pH 7.5 and heating it at 37C with 850 rpm ended up working to elute the nonspecific DNA while retaining my biotinylated DNA.
Halo. Jacob Perkins, I'm glad to hear that you were able to resolve the issue with the nonspecific binding of DNA to biotinylated beads! It's great that you were able to identify the specific problem with pH and heating, and that you found a solution that worked for your probe. Optimizing the elution conditions, such as pH and temperature, is an important step in reducing nonspecific binding and improving the specificity of your assay. Best of luck with your experiments!
Nonspecific binding of DNA to biotinylated beads can be a common problem in various experiments, including chromatin immunoprecipitation (ChIP) assays and DNA pull-down assays. Here are some steps you can take to reduce nonspecific binding:
By following these steps, you can minimize the nonspecific binding of DNA to biotinylated beads and increase the specificity of your assay.
Best regards MJ
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