Methods Used for Measuring Radioactivity

The following points highlight the top two methods of measuring radioactivity. The methods are: 1. Scintillation Counting 2. Autoradiography.

Measuring Radioactivity: Method # 1.

Scintillation Counting:

If a sample is in liquid or on a strip of filter paper, the amount of radioactivity is measured using scintillation counting. If the sample is flat, such as an agarose gel, scientists use au­toradiography to pinpoint the location of ra­dioactive bands or spots.

Scintillation count­ing relies on special chemicals called scintillates. These emit a flash of light when high energy electrons known as beta-particles are released by radioactive isotopes in DNA. The light pulses from the scintillant are de­tected by a photocell (Fig 3.7).

A scintillation counter is simply a very sensitive device for recording faint light pulses. To use the scintil­lation counter, radioactive samples to be mea­sured are added to a vial containing scintillant fluid and loaded into the counter.

The counter prints out the number of light flashes it de­tects within a designated time. The researcher can then compare the number of flashes with a series of standards to determine relative amount of radioactive DNA in the sample.

Scintillation counter can be used to mea­sure light generated by chemical reactions also. In this case, the light is emitted directly and so no scintillant fluid is needed and lumines­cent sample is merely inserted directly.

Measuring Radioactivity: Method # 2.

Autoradiography:

This is used for detecting radioactively labelled DNA or RNA in a gel after separation by elec­trophoresis. Very often DNA or RNA bands in a gel are transferred onto membranes by blot­ting to allow more convenient handling dur­ing autoradiography. In addition, autoradio­graphy can detect radioactive DNA bound to filter paper during hybridization experiment.

Whether the radioactive DNA is in a gel or on a membrane or on a piece of filter paper, the radioactive isotope emits beta-particles. The particles will turn regular photographic film black, exactly the same way that light turns photographic film black.

To do autorad­iography, the gel or filter containing radioac­tive DNA is dried so that photographic film does not stick. Next, a sheet of photographic film is laid on top of the gel or filter and left for several hours or sometimes even for days. The film darkens where the radioactive DNA bands or spots are found (Fig. 3.8). Exposing the film must be carried out in a dark room to avoid visible light.