Autoradiography

Autoradiography of biomolecules

Autoradiography is a class of techniques in which biomolecules are labelled with radioisotopes, such that their presence can be detected in an autoradiogram ("self-picture"). Radioisotopes such as ¹⁴C, ³²P, and ³⁵S emit radiation that exposes photographic or X-Ray film. Substitution of ³²P into the phosphate backbone of DNA, or ³⁵S into the sulfur-containing amino acids of protein allows labelled molecules to be detected.

In the particular experiment shown, a DNA sequencing gel is dried onto filter paper, the paper is placed inside a light-proof X-Ray cassette box (left), and overlain with a sheet of X-ray film. The cassette is sealed shut and the exposure left for several hours to several days. The film records the positions of the radio-labelled DNA termination fragments. In the example, the DNA sequencing "ladder" is read directly from the autoradiogram.

In other applications, a radio-isotope labelled Southern Blot filter DNA exposes the film, which when developed shows a pattern of black bands that indicate the presence or absence, and the sizes of labelled DNA in the blot. Alignment marks in three of the four corners are made with radio-luminescent ink that also exposes the film. The blot can be aligned with the film by means of the three index marks. [With three marks, only one alignment is possible] In related experiments, the alignment can be used to locate particular DNA bands at precise positions on the blot, which can then be recovered for further experiments.