Question

You want to know if a culture of cells is in the process of DNA synthesis. You incubate your cells in the presence of radioactive thymidine to see if it is being incorporated into the DNA. What is the best technique to detect the labeled deoxynucleotide in nuclear DNA?

Answer 1

To detect new DNA synthesis, cells are incubated with radioactive thymidine followed by autoradiography to visualize the incorporation of the radioactive label into newly synthesized DNA, identifying the cells in the S-phase of the cell cycle.

Step-by-step explanation:

Detecting Radioactively Labeled DNA

To determine if a culture of cells is in the process of DNA synthesis, radioactive thymidine can be used. The cells are incubated with ³H-thymine, which is incorporated into DNA as thymidine triphosphate (dTTP). After incorporation, the detection of the labeled deoxynucleotide can be accomplished using a technique called autoradiography.

Autoradiography involves dipping the prepared slides of the cells in a light-sensitive emulsion, similar to that found in film, which captures the decay of the radioactive label and creates a visual representation of DNA synthesis on the film after exposure and development. Samples can be chased with unlabeled thymine to follow incorporation over time, and results can be analyzed to determine periods of DNA synthesis in the cell cycle, including the S-phase. Incorrect application of radioactive labels to protein instead of DNA, such as using radioactive sulfur for proteins and radioactive phosphorus for DNA, should be avoided to ensure accurate results.