I'm reading some papers for the first generation sequencing methods and some earlier than them, like Ray's Wu first time DNA sequencing from a λ phage virus cohesive 5' ends. Ray Wu, like Maxam-Gilbert and Sanger, used 32P / 3H to label either dNTPs or DNA molecules.

But what happens if you add such radioactive materials inside a test tube with DNA and other molecules like DNA Pol I? For example, 32P emits electrons at 1.709 MeV. Couldn't these high energy electrons harm the DNA or Pol I molecules (bonding destruction), that are present nearby at the time of reaction?


Experimentalists pretend, or act as if, those effects are negligible, or miniscule. One could calculate the number of DNA or protein molecules that are affected, by plugging in the specific activity of the dNTPs, the concentration of the potential targets, the volume of the reaction, and the time.

Certainly if you create radiolabeled nucleic acids and store them, then over time those strands will undergo scission as the phosphorous emits it's beta particle. Some of the neighbouring bases might be hit.

If you pulse label growing bacteria or phage you can then mutagenize and select for mutants in interesting pathways (this was called radiosuicide and was invented by Clarence Fuerst).

It is also possible to label bacteria and feed them to C. elegans such that the progeny of those worms will contain new mendelian mutations induced by the radioactive DNA in their mothers' germline.

  • $\begingroup$ Thank you very much. Your answer is very informative. I liked the use of "pretend" verb. Do you have any paper in mind "counting" the destroyed molecules from such chemical agents? $\endgroup$ – F.N Oct 25 '16 at 18:00

It's worth considering how many high energy electrons are actually released compared to the number of molecules in the tube. For instance, I routinely work with $^{32}P$-labeled DNA, and measure the specific activity of samples by scintillation counting. For me, a very hot stock sample of labeled DNA (~1000x more concentrated than I would use in an experiment) will register on the order of $10^8$ disintegrations per minute (dpm). That means each minute, about $10^8$ beta particles are emitted by decaying phosphorous atoms inside the sample. But the amount of DNA molecules in that same tube is about 50 pmol, or about $3 \times 10^{14}$ molecules.

So if each of those beta particles were absorbed by a DNA molecule in the same sample, thus damaging it, that would be a rate of self-damage of $10^{-6} min^{-1}$. Of course, most of those beta particles actually make it out of the tube, so the actual rate of self-damage is much lower still. Since we're probably interested in measuring processes on much shorter time scales than this self-damage rate, it becomes negligible.

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    $\begingroup$ Additionally, in an experiment you are generally not looking at the properties of a single molecule. If you were sequencing this DNA, for example, it is exceedingly unlikely, especially given the calculations in your answer, that the majority of bases at a single position would be mutated and therefore affect the result. $\endgroup$ – canadianer Jan 17 at 19:25

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