Application:
• Cairns’ technique for measuring the length of DNA molecules by autoradiography
Autoradiography
- Cells are grown in a solution containing radioactive thymidine (tritiated thymidine – 3H-T)
- The tritiated thymidine is incorporated into the chromosomal DNA of the cell (3H-T is used as thymidine is not present in RNA)
- The chromosomes are isolated by gently lysing the cells and fixing the chromosomes to a photographic surface
- The surface is then immersed in a radioactively-sensitive emulsion containing silver bromide (AgBr)
- The radiation released from the tritiated thymidine converts the Ag+ ions in silver bromide into insoluble metal grains
- Following a period of exposure, excess silver bromide is washed away, leaving the silver grains to appear as small black dots
- When the photographic film is developed, the chromosomal DNA can be visualised with an electron microscope
Summary of the Process of Autoradiography
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Chromosome Length
- John Cairns pioneered a technique for measuring the length of DNA molecules by autoradiography
- Previously, chromosome length could only be measured while condensed during mitosis (very inaccurate due to supercoiling)
- Cairns used autoradiography to visualise the chromosomes whilst uncoiled, allowing for more accurate indications of length
- By using tritiated uracil (3H-U), regions of active transcription can be identified within the uncoiled chromosome
Uncoiled Chromosomes Identified with Autoradiography
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Other Discoveries
John Cairns was further able to use autoradiography to demonstrate key events which occur during chromosomal replication
- DNA replication involves formation of a replication bubble (and prokaryotic replication involves a single origin of replication)
- DNA replication is bi-directional (it occurs independently at both ends of the replication bubble)
1. Evidence for the Formation of Replication Bubbles (Prokaryotes)
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2. Evidence that Replication is Bi-Directional
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Interview with John Cairns about Autoradiography
Excerpt part of the Oral History Collection of the CSHL Digital Archives