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How do chemists separate single nucleotides from DNA strands? How do they add those nucleotides to e.g. a PCR reaction in order to replicate DNA?

I ask this because in my microbiology class, my teacher didn't know the exact answer.

Do you cleave off nucleotides by hydrolysis? Do you somehow build nucleotides?

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  • $\begingroup$ I'm not sure the exact process but genomes are deconstructed and reconstructed using enzymes such as taq polymerase, reverse transcriptase and the correct primers ( which target the specific site that you want the enzyme to attach to the dna strand) . Techniques such as polymerase chain reaction, electrophoresis and lots of bio processing would be used along with other techniques I'm not aware of. I'm not sure how a new sequence is added to a genome there would be too many variables to cover in a practical answer ! $\endgroup$ – Technetium Oct 6 '15 at 3:46
  • $\begingroup$ When I did PCR and electrophoresis I added the component molecules to the mix like a primordial soup . The nucleotide solution was from sigma aldrich I think. Can't remember if they were synthetic or not. The enzyme uses them to build the new strand. In the PPE reaction the DNA denatures at a certain heat. $\endgroup$ – Technetium Oct 6 '15 at 4:07
  • $\begingroup$ I’m not sure what you’re asking. Are you interested in where the nucleotides that one adds to PCR reactions are acquired from? $\endgroup$ – Jan Oct 7 '15 at 14:35
  • $\begingroup$ There's a few technical questions in there. $\endgroup$ – Technetium Oct 7 '15 at 14:47
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You just buy the nucleoside triphosphates from your favorite supplier, they're commercially available.

As far as I know the NTPs you can buy are still produced biologically and not synthesized (see this example from Sigma Aldrich, which states a microbial source). So you grow a large amount of bacteria, lyse the cells to get at the contents and then digest the DNA or RNA with an appropriate enzyme that cuts it into single nucleotides. To get the triphosphate from that you have to phosphorylate the monophosphates again with the appropriate kinase.

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  • $\begingroup$ They have recently discovered that synthetic nucleotides work the same and a much less expensive to produce in high yields. $\endgroup$ – Technetium Oct 7 '15 at 1:15
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According to sigma aldrich the nucleotides used for PCR are now produced synthetically.

The low yield and high cost to produce them biological makes it impractical. When produced synthetically they get a high yield at low cost.

They are added to the pcr mixture in a pre prepared solution and usually transferred via micropippette or a machine.

The new gene can be built from these nucleotides.

Using gene therapy techniques the new DNA can be incorporated into a genome.

One, of multiple techniques, uses an adenovirus vector with the new DNA packaged inside to transport and insert the new DNA to the cell in the form of a nucleic acid polymer which incorporates the genes into the chromosomes.

Experiments were done to compare the efficiency of the synthetic genes to the biological ones with the need for biological nucleotides as a control. Now they know the gene built from synthetic nucleotides shows the same expression as the biological gene of the same code synthetic nucleotides are used. However microbial produced nucleotides can still be used.

Ref:Wikipedia, Gene therapy. Sigma aldrich - Technical support ph - 1800 800 097

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  • $\begingroup$ Sigma sells multiple different versions, some declared as "from microbial sources" and others without a source, which are likely the synthetic ones. There doesn't seem to be a huge price difference. $\endgroup$ – Mad Scientist Oct 7 '15 at 6:47
  • $\begingroup$ I called and asked them . Your free to do the same. $\endgroup$ – Technetium Oct 7 '15 at 7:37
  • $\begingroup$ I'm not saying the microbial sourced ones don't exist. $\endgroup$ – Technetium Oct 7 '15 at 7:41

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