How does Cairns’ technique use autoradiography to measure the length of DNA?

SouravNovember 8, 2024

Answer

Cairns’ technique for measuring the length of DNA utilizes autoradiography, a method that allows for the visualization of DNA molecules in a way that provides accurate measurements of their length. Here’s a detailed overview of how this technique works and its significance:

Overview of Cairns’ Technique Using Autoradiography

1. Incorporation of Radioactive Thymidine:
   • Cairns began by culturing Escherichia coli (E. coli) cells in a medium containing tritiated thymidine (³H-thymidine), a radioactive form of thymidine. This radioactive compound is incorporated into the bacterial DNA during replication, effectively labeling the DNA.
2. Cell Lysis and Chromosome Isolation:
   • After allowing sufficient time for incorporation, the cells are gently lysed to release the chromosomal DNA. This step is crucial as it minimizes damage to the DNA structure, preserving its integrity for measurement.
3. Preparation for Autoradiography:
   • The isolated DNA is then fixed onto a photographic surface or membrane, which is coated with a radioactively sensitive emulsion containing silver bromide (AgBr). This setup is essential for capturing the radiation emitted by the incorporated tritiated thymidine.
4. Exposure and Development:
   • The photographic surface is exposed for an extended period to allow the radiation from the radioactive thymidine to convert Ag⁺ ions in silver bromide into insoluble metallic silver grains. After exposure, excess unexposed silver bromide is washed away, leaving behind a pattern of black dots corresponding to the radioactive emissions.
5. Visualization and Measurement:
   • The developed autoradiographs can be examined using an electron microscope, revealing tracks of silver grains that represent the length of the DNA molecules. Cairns measured these tracks to determine the lengths of the circular chromosomes in E. coli.

Significance of Cairns’ Technique

• Accurate Measurement of DNA Length:
  • Prior to Cairns’ work, chromosome lengths were typically measured during mitosis when chromosomes are condensed, leading to inaccuracies due to supercoiling. By visualizing uncoiled chromosomes, Cairns provided more accurate measurements of DNA length.
• Insights into DNA Replication:
  • The autoradiographs also allowed Cairns to observe key events in DNA replication, including the formation of replication bubbles and bi-directional replication processes in prokaryotes.
• Establishment of Circular Chromosomes:
  • Cairns’ technique was pivotal in demonstrating that bacterial chromosomes are circular, which was an important discovery in understanding prokaryotic genetics.