32P labelling

32P

³²P-labelling of DNA

For use in autoradiography, especially as applied to DNA sequencing reaction before the advent of fluorescent labeling techniques, a radioactively-labelled dNTP molecule is synthesized with the first ("alpha") PO₄containing radioactive ³²P rather than non-radioactive ³³P. During synthesis of the new DNA strand, the outer two PO₄ groups are cleaved off and the labelled PO₄is incorporated into the DNA backbone.

An alternative method of labeling substitutes ³⁵S for the ester-bonded O atom on the alpha PO₄ and leaves the P unlabelled. This has the advantage of a longer half-life that allows longer exposures. It is also less radio-energetic so as to leave finer lines in the DNA ladder (it doesn't penetrate the film as well). This also makes ³⁵S safer to handle.

NB: Technical Note: Both radio-isotopes are β (Beta)-emitters. ³²P has a radioactive half-life of 14.2 days, and breaks the PO₄ group when it decays, such that it is not available for the labeling reaction. This means that it is necessary to increase the specific activity of the labeling reaction over time, for example twice as much label needs to be added when it is one half-life (14.2 days) old to get the same signal strength. In practice, new label needs to be ordered once a month. ³⁵S has a half-life of 87.4 days (six times that of ³²P), and being linked to the alpha-PO₄, does not remove it on decay. The good news is that specific activity of the label need not be adjusted over time. The bad news is that you couldn't anyway, because a decayed molecule participates in the labeling reaction just as well as the radioactive label.