[DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

Scheme 20 is nice it adds an acyl group. Rutgers 2014 igem project used an acyl group to protect dNTPs. They used ammonia to remove it. I believe ammonia does not require protecting groups on nucleotide bases in fact it's sometimes used to remove them. However scheme 20 only works for bases a and t. I wonder if you could modify the enzyme to work on c and g. Scheme 17 only works on a u base.

Well I've thought about this at length actually. Two posable strategies for alternative blocking groups have occurred to me. The first it may, just may, be posable to use a disaccharide synthase to add a sugar based compound to the 3 prime position of a dNTP. You can see scheme 31 in that paper shows a way of using a disaccharide synthase to add at the 5 prime position but there are a lot of disaccharides in nature and lots of different enzymes to make them. You'd have to screen a lot of disaccharide synthases till you found one with week activity for 3 prime addition then do directed evolution to get something that could give you dNTPs blocked by joining another monosaccharide to the ribose. To the best of my knowledge all disaccharides break down into monosaccharides in sufficiently hot water. So you could make your own thermal liable blocked dNTP. It won't be cheeper to do this than buy cleanamp.

The other option might be to use dirty ends. When dna breaks it doesn't always leave clean 3 prime ends. Often you get a lot of 3 prime ends with PO4H2 groups on them. You can't polymerise off this. But there is an enzyme PNKP that nips off the PO4H2 group so polymerisation can continue. If you used dirty dNTPs and used PNKP instead of heat to deblock that might be an option. However the only simple way I can think of to get dirty dNTPs is to get a DNA sample, subject it to lots of oxygen, possibly ozone. Use exonucleases to break it down into clean and dirty dNMPs painstakingly purify out the dirty dNMPs turn them into dirty dNTPs using NDP / NMP kinases then separate them by base. This will also not be cheeper or easier than buying cleanamp.

So the question in my mind is how hard would it be to make a dirty dNTP chemically with out an enzyme. Is there a reagent I could add to dNTPs and phosphoric acid that would attach PO4H2 to the 3 prime location with out the base or 5 prime end needing protecting groups. There are 4 OH groups hanging off the phosphors at the 5 prime end so can we expect some PO4H2 groups added here? What if I turned the phosphoric acid into a free radical before adding it using electrical discharge? All things I've considered. Basically I'm a sucky chemist and it takes a very talented chemist to do serious organic synthesis only using reagents you can purify out of things you can buy at your local supermarket or buy from amazon.

And again none of this is going to be cheeper or easier than cleanamp. But as I said a cheep solution is unrealistic this method is aimed at those who a) want a learning experience or b) can't get industry made DNA sequences.

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