Q.In an experiment, E. coli is grown in a medium containing 14NH4Cl. (14N is the light isotope of Nitrogen) followed by growing it for six generations in a medium having heavy isotope of nitrogen (15N). After six generations, their DNA was extracted and subjected to CsCl density gradient centrifugation. Identify the correct density (Light/Hybrid/Heavy) and ratio of the bands of DNA in CsCl density gradient centrifugation. (A) Hybrid : Heavy, 1 : 16 (B) Light : Heavy, 1 : 31 (C) Hybrid : Heavy, 1 : 31 (D) Light : Heavy, 1 : 05
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Start your 14-day free trial to unlock the full solution →After six generations of semi-conservative DNA replication in a heavy nitrogen medium, the original two light strands will always form hybrid DNA, while all other DNA will be heavy. This results in a Hybrid : Heavy DNA ratio of 1 : 31.
The core concept here is the semi-conservative nature of DNA replication, famously demonstrated by the Meselson-Stahl experiment. When DNA replicates, each new double helix consists of one original (parental) strand and one newly synthesized strand. This principle dictates how the nitrogen isotopes are distributed in the DNA molecules over successive generations, which in turn affects their density.
Nitrogen is a key component of DNA bases.
- 14N is the common, "light" isotope of nitrogen.
- 15N is a heavier isotope. DNA containing 15N will be denser than DNA containing 14N. DNA with one 14N strand and one 15N strand (a hybrid molecule) will have an intermediate density.
CsCl density gradient centrifugation separates molecules based on their density. When DNA is spun in a CsCl solution, it forms bands at positions where its density matches the density of the CsCl solution.
- Light DNA (14N/14N) will form a band at the highest position (least dense).
- Hybrid DNA (14N/15N) will form a band at an intermediate position.
- Heavy DNA (15N/15N) will form a band at the lowest position (most dense).
Let's trace the DNA composition over the generations:
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Initial State (Generation 0):
The E. coli is initially grown in a medium containing 14NH4Cl. This means all the nitrogen incorporated into their DNA is the light isotope, 14N.
So, all DNA molecules are 14N/14N (Light).
Let's assume we start with 1 DNA molecule. It has two 14N strands.
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First Generation (after 1 replication in 15N medium):
The E. coli is transferred to a medium containing 15NH4Cl and allowed to replicate once.
According to semi-conservative replication, the original 14N/14N DNA molecule unwinds. Each 14N strand serves as a template for a new strand synthesized using 15N.
This results in two DNA molecules, each consisting of one 14N strand and one 15N strand. These are Hybrid DNA molecules.
- Total DNA molecules: 2^1 = 2
- Hybrid DNA molecules: 2
- Heavy DNA molecules: 0
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Second Generation (after 2 replications in 15N medium):
The two hybrid DNA molecules from the first generation replicate again in the 15N medium.
Each hybrid molecule (14N/15N) unwinds.
- The 14N strand acts as a template, pairing with a new 15N strand to form a new Hybrid molecule (14N/15N).
- The 15N strand acts as a template, pairing with a new 15N strand to form a new Heavy molecule (15N/15N). Since there were 2 hybrid molecules, they will produce 2 hybrid and 2 heavy molecules.
- Total DNA molecules: 2^2 = 4
- Hybrid DNA molecules: 2
- Heavy DNA molecules: 2
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Generalizing for 'n' Generations:
Notice a pattern:
- The two original 14N strands (from the very first DNA molecule) will always serve as templates. Each time they replicate in the 15N medium, they will pair with a newly synthesized 15N strand. This means there will always be exactly 2 Hybrid DNA molecules, regardless of how many generations pass, as long as the replication continues in the 15N medium. …
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