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Phosphorothioate group

Scintillation counters measure radioactivity in liquid samples whereas autoradiography is used to locate radioactive molecules on gels or membranes.

photocell (Fig. 21.20). 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 the relative amount of radioactive DNA in the sample.

Scintillation counters can be used to measure light generated by chemical reactions, also. In this case, the light is emitted directly so no scintillant fluid is needed and the luminescent sample is merely inserted directly. The detection of light emitted by luciferase and lumi-phos in genetic analysis is described in Ch. 25.

Autoradiography is used for detecting radioactively labeled 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 autora-diography. In addition, autoradiography can detect radioactive DNA bound to filter paper during hybridization experiments. Whether the radioactive DNA is in a gel or on a membrane or 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 the 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. 21.21). Exposing the film must be carried out in a dark room to avoid visible light.

scintillation counter Machine that detects and counts pulses of light

Fluorescence in the Detection of DNA and RNA 585

FIGURE 21.20 Scintillation Counter is Used to Measure Radioactivity

Radioactive DNA is mixed with a liquid scintillant. The scintillant molecules absorb the b-particles emitted by the 32P in the DNA, and in turn emit a flash of light. The photocell counts the number of light pulses in a specific time period.

Sample

Light

Sample

Light

Counter

Radioactive DNA

molecule b-particle

Counter

Autoradiograph

Gel with radioactive but invisible bands of DNA

Lay film on gel and keep in dark, then develop film

Film

Film shows position of bands

FIGURE 21.21 Autoradiography to Detect Radio-Labeled DNA or RNA

A gel containing radioactive DNA or RNA is dried and a piece of photographic film is laid over the top. The two are loaded into a cassette case that prevents light from entering. After some time (hours to days), the film is developed and dark lines appear where the radioactive DNA was present.

DNA or RNA may be labeled with fluorescent dyes.

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