Meselson And Stahl Experiment on DNA Replication

History of the Experiment

The Meselson and Stahl experiment, conducted in 1958, is a classic in molecular biology that provided unequivocal proof for the semi-conservative mechanism of DNA replication. The experiment gave a clear view of how DNA strands separate and become templates for new strands. It nailed down the basics of how we understand genetics in terms of inheritance and how their materials are to be replicated.

Concept of DNA Replication

Replication of DNA is a process wherein one double-stranded DNA molecule is copied to replicate two identical DNA molecules. This process lies at the core of cell division, as through this, one generation can pass genetic material to succeeding generations. The Meselson-Stahl experiment tested models of DNA replication that had been proposed: the conservative, semi-conservative, and dispersive. Even the central dogma of molecular biology indicates that DNA replicates.

Discovery of DNA Structure

In 1953, James Watson and Francis Crick discovered and gave the feature of the double helix structure of DNA, making explicit the molecular basis of genetic inheritance. Their model indicated that each strand of the DNA double helix could act as a template for generating a new complementary strand. It gave indications for the existence of a semi-conservative mechanism for DNA replication.

DNA Replication Hypotheses

There were three models put forward to account for how DNA replicates:

• Conservative Model: In this model, the original DNA molecule remains intact, and an entirely new molecule is synthesised.

• Semi-Conservative Model: Each of the two new DNA molecules would contain one old strand and one newly synthesised strand.

• Dispersive Model: Here the original DNA molecule breaks into pieces, and new DNA is synthesized in a dispersed pattern mixing old and new DNA segments.

The Scientists: Meselson and Stahl

Matthew Meselson: This American geneticist and molecular biologist was born in 1930. His work on the points of DNA replication and repair mechanisms brought our knowledge about them a long way forward.

Franklin Stahl: An American geneticist born in 1929, Stahl is also one of the leading investigators in molecular biology and in genetic recombination and DNA replication.

Roles and Contributions

The Meselson and Stahl collaboration on the DNA replication experiment radicalised molecular biology by providing solid experimental proof for the semiconservative model of DNA replication. Their work established certain methodologies that later proved to be milestone procedures for future studies in genetics.

Experimental Design of Meselson and Stahl Experiment

The main objective of the experiment by Meselson and Stahl was to ascertain the correct model of DNA replication through differentiation between the conservative, semiconservative, and dispersive models.

Materials And Methods

The reason for choosing E. coli is because of its fast growth rate and well-known genetics. Hence, it would provide a very good model organism in the DNA replication study.

Significance of using Nitrogen Isotopes (15N and 14N)

Meselson and Stahl used the heavy nitrogen (15N) and light nitrogen (14N) isotopes to label the DNA. First, E. coli bacteria were grown in 15N medium to ensure that all DNA in the bacteria contained this heavy isotope. Then, transferring them to a 14N medium allowed them to trace how the lighter isotope entered newly replicated DNA strands.

Step-Wise Procedure

The step-wise breakdown on how Meselson and Stahl did their experiment to prove that DNA replication is semi-conservative is given below-

1. Culture of E. Coli In 15N :

E. coli were grown for several generations on a medium with 15N so that all its DNA was labelled with the heavy nitrogen isotope.

2. Shifting to 14N Medium:

The bacteria were transferred into a medium containing 14N. At different time intervals, samples of DNA were withdrawn to monitor the incorporation of the lighter isotope.

3. Extraction and Analysis of DNA Samples:

DNA was isolated from the bacterial cells in density gradient centrifugation, a method that separates DNA according to its density.

Diagram: Meselson And Stahl's Experiment

The diagram given below shows the process of Meselson and Stahl's experiment:

Results of the Experiment

The results of the experiment are as follows:

Generation 0 (Heavy DNA)

After growing in the 15N medium, E. coli DNA was extracted and run through the density gradient, resolved into a single discrete band, which corresponded to heavy DNA (15N-15N).

Generation 1 (Hybrid DNA)

After one replication cycle on a 14N medium, the gradient contained a single band of intermediate density, characteristic of hybrid DNA. This was inconsistent with the conservative model of replication by which two separate bands should have appeared, one heavy and one light.

Subsequent Generations

After two rounds of replication, two bands appeared: one at the middle position and another at the light DNA position (14N-14N). The result conformed to the semiconservative model. That is, every new DNA molecule contains one old strand and one new strand.

MCQs on Messelson and Stahl Experiment

Q1. Assertion [A]: The nature of DNA replication is semiconservative.

Reason [R]: Newly formed DNA contains one daughter and one parent DNA

Option 1: Both, A and R, are correct and R explains A

Option 2: Both, A and R, are correct but R not explains A

Option 3: A is correct but R is wrong

Option 4: A is wrong but R is correct

Correct answer: 1) Both, A and R, are correct and R explains A

Explanation:

DNA replication is categorized as "semiconservative" because parental DNA strands are passed to newly produced daughter DNA helices, while the other strand appears to be complementary to DNA. DNA replication is also "semi-discontinuous". One strand of DNA is continuously developed whereas complementary DNA is discontinuously formed through Okazaki fragment

Hence, the correct answer is Option 1) Both, A and R, are correct and R explains A

Q2. Components used in Meselson and Stahl's experiment

Option 1: Heavy Nitrogen

Option 2: Radioactive Nitrogen

Option 3: CsCl (Cesium Chloride)

Option 4: Any two of them

Correct answer: (4) Any two of them.

Explanation:

Experiments done by Meselson and Stahl -

They grew E.Coli in a medium containing ( is the heavy isotope of nitrogen). As a result was incorporated into newly synthesized normal DNA by centrifugation in a cesium chloride () density gradient.

Hence, the correct answer is Option (4) Any two of them.

Q3. The method developed by Meselson and Stahl to separate heavy DNA with N’s from DNA N’4, for providing evidence for semi-conservative replication of DNA is

Option 1: Ion exchange chromatography

Option 2: Density gradient centrifugation

Option 3: Isopycnic centrifugation

Option 4: Gel filtration

Correct answer: 2) Density gradient centrifugation

Explanation:

Experiments done by Meselson and Stahl -

They transferred the E. coli bacteria Cells into a medium with normal 14NH4Cl samples and extracted DNA Various samples were separated independently on CsSl gradients to measure. the deities of DNA.

Hence, the correct option is 2) Density gradient centrifugation

Also read-

• Practice MCQs on Replication of DNA

• RNA Processing

• RNA Splicing