Given below is a list of steps Meselson and Stahl carried out in their experiment to prove that DNA replication is semi-conservative. Select the option that gives the correct sequence of steps followed by them.(i) Bacteria transferred to a N¹⁴ medium and sampled every 20 minutes.(ii) All bacteria contain hybrid DNA (N¹⁴ DNA and N¹⁵DNA).(iii) Bacteria grown in N¹⁵ medium for many generations.(iv) All bacteria contain N¹⁵ DNA.(v) Bacteria contain either all N¹⁴ DNA or all hybrid DNA.

The correct option is (iii) → (iv) → (i) → (ii) → (v).

Step-by-Step Explanation of the Meselson-Stahl Experiment:

1. Growing bacteria in heavy isotope medium (Step iii):
Matthew Meselson and Franklin Stahl began their experiment by growing Escherichia coli (E. coli) bacteria in a culture medium containing heavy nitrogen (¹⁵N) in the form of ammonium chloride (¹⁵NH₄Cl) for many generations. ¹⁵N is a heavy isotope of nitrogen, but it is not radioactive.

2. Incorporation of ¹⁵N into DNA (Step iv):
Because the bacteria were grown in the ¹⁵N medium for many generations, the heavy nitrogen isotope was successfully incorporated into the nitrogenous bases of the bacterial DNA. At this stage, all the bacteria contained DNA that was completely labeled with heavy nitrogen (all ¹⁵N DNA).

3. Transfer of bacteria to light isotope medium and sampling (Step i):
Next, the bacteria were transferred to a fresh medium containing normal light nitrogen (¹⁴NH₄Cl) as the sole nitrogen source. Cell samples were collected at regular intervals of 20 minutes (which is the doubling or generation time of E. coli) to extract and analyze the DNA density using cesium chloride (CsCl) density gradient centrifugation.

The generation time can be represented by the formula for the number of cells after n generations:
$$N=N_0\times 2^n$$
where N₀ is the initial number of cells, and each generation n corresponds to a 20-minute interval.

4. Formation of hybrid DNA after one generation (Step ii):
After exactly one generation (20 minutes), the DNA extracted from the culture showed an intermediate density. This is because each newly formed DNA molecule contained one parental strand (heavy, ¹⁵N) and one newly synthesized strand (light, ¹⁴N), forming hybrid (¹⁴N-¹⁵N) DNA. At this point, 100% of the bacteria contained hybrid DNA.

5. Appearance of light and hybrid DNA in subsequent generations (Step v):
After two generations (40 minutes), the DNA extracted from the culture was composed of equal amounts of hybrid DNA (¹⁴N-¹⁵N) and light DNA (completely ¹⁴N-¹⁴N). Thus, the bacteria contained either all ¹⁴N DNA or hybrid DNA. This result conclusively proved that DNA replication is semi-conservative.