Q.Answer the following questions based on Meselson and Stahl’s experiment on E. coli :
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Start your 14-day free trial to unlock the full solution →Meselson and Stahl used nitrogen-15 and nitrogen-14 isotopes to label DNA across generations, then separated heavy from light DNA by cesium chloride density gradient centrifugation, proving that DNA replication is semiconservative.
The Meselson-Stahl experiment stands as one of the most elegant demonstrations in molecular biology, designed to settle a fundamental question: how does DNA copy itself? By 1958, Watson and Crick had proposed the double helix structure, but three models of replication competed for acceptance—conservative (the original helix stays intact, a completely new copy forms), semiconservative (each strand serves as a template, so every new DNA molecule contains one old and one new strand), and dispersive (old and new DNA segments intersperse). Matthew Meselson and Franklin Stahl devised a brilliantly simple experiment using isotopes of nitrogen to track DNA across bacterial generations.
(a) The nitrogen source
The chemical substance they used was ammonium chloride (NH₄Cl) containing the heavy isotope nitrogen-15 (¹⁵N). Nitrogen is a key component of all nitrogenous bases in DNA—adenine, guanine, cytosine, and thymine—so every new DNA molecule synthesized by the bacterium would incorporate whichever isotope was available in the growth medium. By controlling the nitrogen source, Meselson and Stahl could label the DNA and track it through successive rounds of replication.
(b) Why synthesize both heavy and light DNA?
The researchers needed to create a starting population of bacteria with fully heavy DNA, then watch what happened when those cells replicated in a medium containing only the normal, light isotope nitrogen-14 (¹⁴N). Here's the logic:
- They first grew E. coli for many generations in a medium with ¹⁵N as the sole nitrogen source. Every nitrogenous base incorporated the heavy isotope, so the entire DNA became "heavy" (¹⁵N-¹⁵N).
- Then they transferred these bacteria to a medium containing only ¹⁴N (light nitrogen). As the cells divided, any newly synthesized DNA strand would incorporate the light isotope.
- By examining the density of DNA after one generation, two generations, and so on, they could distinguish between the three replication models. If replication were semiconservative, the first generation should produce hybrid DNA (one heavy strand, one light strand). If conservative, the first generation should show separate heavy and light DNA molecules with no hybrids.
The synthesis of both types allowed them to create a "before and after" scenario, tracking the fate of the original heavy strands as new light strands were made.
(c) Distinguishing heavy from light DNA
Meselson and Stahl used cesium chloride (CsCl) density gradient centrifugation, a technique that separates molecules based on their buoyant density. Here's how it worked:
They dissolved cesium chloride in a solution and spun it at extremely high speeds (around 140,000 times gravity) for many hours. The cesium ions formed a smooth density gradient in the centrifuge tube—denser at the bottom, lighter at the top. When DNA was added to this gradient and centrifuged, each DNA molecule settled at the point in the gradient where its density matched the surrounding cesium chloride solution.
Heavy DNA (¹⁵N-¹⁵N) is denser than light DNA (¹⁴N-¹⁴N) because nitrogen-15 has one extra neutron per atom. Hybrid DNA (¹⁵N-¹⁴N) has an intermediate density, settling between the two.
After centrifugation, they could see distinct bands in the tube:
- DNA from bacteria grown only in ¹⁵N medium formed a band low in the tube (heavy).
- DNA from bacteria grown only in ¹⁴N medium formed a band higher up (light). …
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