I would require more information to answer this question effectively.

So one of your primers has been synthesized including the 5'-thiol-C6 modified base at the 5' end of the primer sequence? Is that correct?

@ Zachery

Yes, that is correct.

Firstly, it is possible there is formation of disulfide bonds between the oligos preventing the maleimide from reacting. I would recommend TCEP (Tris[2-carboxyethyl] phosphine) instead of DTT as it can be difficult to remove all traces of DTT from the reduction reaction and even a relatively small amount of free thiol (DTT) can out-compete your oligo for the maleimide reaction since the oligo is likely present at uM concentration. Simply add a 100 X excess (i.e. 30 mM TCEP to 300 uM oligo) of TCEP to a solution of your oligo in reaction buffer, incubate at room temperature for about 2 h, and proceed with your coupling reaction (TCEP is unreactive toward maleimide). Most suppliers of thiol-modified nucleotides recommend that a reduction step be performed and by keeping TCEP in the reaction during coupling it helps to ensure no oxidation occurs.

Second, be sure to include 2 - 4 mM DTT in your PCR reaction, and it's not a bad idea to degas the PCR buffer and oligo mixtures and flush the PCR tube with inert gas before doing the reaction. Thiols can not only oxidize to the disulfide (which is reversible) but easily oxidize to sulfone/sulfoxide/sulfate which are not reversible. Purify the PCR product from the reaction before doing the coupling, phenol/chloroform extraction and precipitation followed by a number of 70% ethanol washings will work or else the use of a commercial PCR product cleanup kit is also an excellent option. Keep 1 - 2 mM TCEP in the buffer you resuspend/elute and store your PCR product in.

Third, perform the reaction in 50 mM phosphate buffer without NaCl at pH 6.5 - 7. Combine the maleimide and thiol PCR product, flush the tube with inert gas, and incubate overnight at room temperature.

Finally, how are you determining if the coupling was successful? A 10kDa PEG molecule is tiny compared to a 1200 bp DNA (molar mass = 780 kDa) so you shouldn't expect to see any size shift on a gel or anything of that nature. Might be the only/best way to determine the degree of modification is by NMR in D2O (with lots of scans since you likely have a very small amount of oligo) and looking for disappearance of the SH signal and/or appearance of the O-CH2-CH2-O signals.

Hope these suggestions help.

First of all,

thanks for your detailed answer that helps me a lot. I have a few questions left:

1. If my SH primer stock solution (in H2O stored@ -20 °C) has gone through a few freeze thaw cycles, is it possible that my SH functionality is already oxidized?

2. Is it better to take an protected primer in order to circumvent the oxization problematics? If so, what primer/protection group would you suggest?

3. Might be a stupid questions, but why no NaCl in the DNA coupling step?

4. For determining DNA coupling the DNA is modified at 3' with an fluorescence dye. After coupling to PEG-maleimide, I prepare hyluronic acid/PEG hydrogel particles. By the fluorescence labeling of the DNA I can qualitatively determine the conjugation of DNA to the hydrogel material. I also have 40k Maleimides available. If I use this one, coupling should be slightly visible on an agarose gel, right?

Many thanks in advance !

Of course, happy to help!

1. I think that freeze/thaw is fine, wouldn't hurt to add a little DTT to the storage solution though. Thiols aren't so reactive that a bit of casual exposure to the air will ruin them, it's just in the PCR reaction where it's being heated to 95 repeatedly that some irreversible oxidation to the sulfone/sulfoxide/sulfate could be an issue.

2. I don't think you need a protected primer, I have used 5'-thiol-C6/maleimide chemistry a few times without difficulty. I don't think it's worth the extra hassle of deprotecting your finished PCR product. Biggest thing is just to remember that even though this is a biochemical reaction we should always take the same precautions we would when doing organic chemistry in terms of using inert atmosphere where appropriate etc.

3. You only need about 5 - 10 mM of a cation to keep the DNA in the double stranded native state, 50 mM buffer is a bit extra but will make sure the pH is steady. 250 mM salt (buffer + NaCl) is quite high. Theoretically it shouldn't interfere but it's always best to keep things as simple as possible. Higher salt neutralizes the negative charge of the DNA and can cause it to form aggregates in solution and do other weird things. Again, shouldn't interfere but why take the risk?

4. Cool method for determining success! It would be difficult to see even a 40k addition by agarose gel electrophoresis, since that would correspond to only roughly a 60bp difference, but if you run a 5% acrylamide/TBE gel you should be able to see the modification, perhaps even your 10k PEG moiety. However, these gels take a bit of practice to get them running perfectly.

Best of luck!