BiologyCBSE Class 12 · Molecular Basis of Inheritance

DNA Replication

Updated 15 October 2026 · Fact-checked

DNA replication is semiconservative: the two strands separate and each serves as a template, so every daughter molecule has one old and one new strand. Meselson and Stahl proved this using 15N in E. coli. At a fork, DNA polymerase builds only 5'→3', so one strand is made continuously and the other in fragments joined by DNA ligase.

Each new DNA molecule keeps one old blue strand, so the hybrid band appears at generation 1 and never disappears.

Illustration drawn for QuestionBuddy.

Understand DNA Replication

The idea. Watson and Crick proposed that the two strands of DNA separate and each acts as a template for a new complementary strand. Each daughter molecule then has one parental and one new strand: semiconservative replication.

Proof: Meselson and Stahl (1958).
1. *E. coli* was grown for many generations in a medium whose only nitrogen source was ** $^{15} N H_{4} C l$ (heavy isotope ¹⁵N), so all its DNA became heavy**.
2. The cells were moved to a medium with normal ** $^{14} N H_{4} C l$ and samples were taken at intervals as they divided (*E. coli* divides about every 20 minutes**).
3. DNA was separated by centrifugation in a caesium chloride (CsCl) density gradient, where heavy, hybrid and light DNA form bands at different positions.
Results:

Generation 0: one heavy band (¹⁵N/¹⁵N).
After 1 generation (20 min): one hybrid (intermediate) band (¹⁵N/¹⁴N).
After 2 generations (40 min): equal amounts of hybrid and light DNA.
After more generations the light band grows, but the hybrid band never disappears: there are always exactly 2 hybrid molecules per original molecule.

This is exactly what the semiconservative model predicts. Taylor and colleagues (1958) obtained similar results in chromosomes of *Vicia faba* using radioactive thymidine.

General rule. Starting from one heavy molecule, after n generations there are $2^{n}$ molecules: 2 hybrid and ** $2^{n} - 2$ light**.

The machinery.

DNA-dependent DNA polymerase is the main enzyme: it uses a DNA template to join deoxynucleotides. It is fast and accurate: *E. coli*, with only 4.6 × 10⁶ bp, completes replication within about 18 minutes, at an average rate of about 2000 bp per second. Mistakes would cause mutations.
Deoxyribonucleoside triphosphates (dNTPs) serve two purposes: they are the substrates and they provide the energy for polymerisation (their two terminal phosphates are high-energy, as in ATP).
Replication fork: the two strands of a long DNA cannot be separated along their entire length at once because that would need a very large amount of energy; replication proceeds within a small opening of the helix, the replication fork.
Polarity: DNA polymerase adds nucleotides only in the 5'→3' direction. So on the template strand with polarity 3'→5' the new strand is made continuously (leading strand), and on the template with polarity 5'→3' it is made discontinuously in short fragments (lagging strand; the fragments are often called Okazaki fragments). DNA ligase joins these fragments.
Origin of replication (ori): replication starts at a definite region, not anywhere. DNA polymerase cannot start replication on its own. This is why, in recombinant DNA work, a piece of DNA must be put into a vector that provides an origin of replication.
In eukaryotes replication happens in the S phase of the cell cycle; it must be coordinated with cell division, since failure of division after replication leads to polyploidy.

Flow chart: origin of replication → strands unwind locally → replication fork → DNA polymerase (5'→3') → leading strand continuous (on 3'→5' template) + lagging strand in fragments (on 5'→3' template) → DNA ligase joins fragments → two semiconservative daughter molecules.

Draw and label 1: replication fork. Checklist: a Y-shaped fork; parental double helix at the stem; the two arms as separated template strands with polarity marked (3'→5' and 5'→3'); on one arm a continuous new strand with an arrow towards the fork; on the other, short fragments with arrows pointing away from the fork. Labels: parental strands, replication fork, template strand (3'→5'), template strand (5'→3'), leading (continuous) strand, lagging (discontinuous) strand, DNA polymerase, DNA ligase, 5' and 3' ends.

Draw and label 2: Meselson-Stahl CsCl bands. Checklist: three centrifuge tubes side by side for generations 0, 1 and 2. Tube 0: one band low (heavy); tube 1: one band in the middle (hybrid); tube 2: two bands, middle (hybrid) and higher (light), equal in thickness. Labels: ¹⁵N/¹⁵N heavy, ¹⁵N/¹⁴N hybrid, ¹⁴N/¹⁴N light, generations.

DNA Replication: key facts

Molecules after n generations: $total = 2^{n}; hybrid = 2; light = 2^{n} - 2$; Starting with one fully ¹⁵N molecule moved to ¹⁴N.
Fractions: $hybrid fraction = \frac{2}{2 ^{n}} = \frac{1}{2 ^{n - 1}}$; n = 1: all hybrid; n = 2: 1/2; n = 3: 1/4; n = 4: 1/8.
Generations from time (E. coli): $n = \frac{time (min)}{20}$; E. coli divides about every 20 minutes.
Direction of synthesis: $new strand: 5^{'} \to 3^{'} only$; Continuous on the 3'→5' template; discontinuous on the 5'→3' template.
Complementary new strand: $3^{'} -TACGGA- 5^{'} \Rightarrow 5^{'} -ATGCCT- 3^{'}$; A pairs with T, G with C; strands are antiparallel.

How to answer DNA Replication questions

Replication questions are either experiments (Meselson-Stahl), mechanism (fork, polarity, enzymes) or calculations of generations and bands.

1For the experiment, give: organism (E. coli), isotopes (¹⁵N then ¹⁴N as NH₄Cl), technique (CsCl density gradient centrifugation), results for generations 0, 1 and 2, and the conclusion (semiconservative).
2For a time-based question, convert time to generations (divide by 20 min), then use 2 hybrid out of $2^{n}$ .
3For the fork, state why a fork (energy to unwind the whole molecule is too high), the 5'→3' rule, which template gives the continuous strand, and the role of ligase.
4For writing a new strand, pair bases (A-T, G-C) and reverse polarity (antiparallel).
5For enzymes, name DNA-dependent DNA polymerase and DNA ligase; mention that polymerase cannot initiate on its own and needs an origin.
6In assertion-reason items on dNTPs, remember their dual role: substrate and energy source.

DNA Replication in real life

The idea, met in an everyday situation. Answer the question in your own words, then compare with the model answer. The one-line hook is what to remember.

Semiconservative replication over several generations

Suppose a student repeats the Meselson-Stahl experiment in a college lab: E. coli fully labelled with ¹⁵N is moved to ¹⁴N medium at 9:00 a.m. and samples are taken at 9:20, 9:40, 10:00 and 10:20 a.m.

Predict the bands in the CsCl gradient for each sample and the fraction of hybrid DNA at 10:20 a.m.

Try it first, then show the solutionSolution

Generations: 20 min = 1, 40 min = 2, 60 min = 3, 80 min = 4.
9:20: all hybrid (1 band). 9:40: hybrid : light = 2 : 2 = 1 : 1. 10:00: 2 hybrid : 6 light = 1 : 3.
10:20: $2^{4}$ = 16 molecules; 2 hybrid and 14 light; hybrid fraction = 2/16 = 1/8 (hybrid : light = 1 : 7).

Answer: Hybrid only, then 1:1, 1:3 and 1:7 hybrid : light; hybrid = 1/8 at 80 minutes.

Memory hook: The two old strands never vanish; they just get diluted.

Tricks to remember DNA Replication

Short memory aids. Say each one aloud once, then use it on a question the same day.

Semi = half old
Each daughter keeps half (one strand) of the parent.
Use it when: Defining semiconservative replication.
Always two hybrids
However many generations, only 2 molecules carry the old ¹⁵N strands.
Use it when: Meselson-Stahl calculations.
Five to three, the only way free
Polymerase builds 5'→3' only; the strand that must go the other way is made in pieces.
Use it when: Continuous vs discontinuous synthesis.

DNA Replication: solved examples

Ordered from board level to HOTS. Try each one before you open the solution.

Example 1: dNTPs' dual role · 1 mark · Very short answerBoard level

Why are deoxyribonucleoside triphosphates said to have a dual role in DNA replication?

Try it first, then show the solutionSolution

They are the substrates (building blocks) added to the new strand, and their two terminal high-energy phosphates provide the energy for polymerisation.

Answer: Substrate and energy source.

Example 2: Why a fork · 3 marks · Short answerBoard level

(a) Why does DNA replication occur within a replication fork and not along the entire length at once? (b) Explain why replication is continuous on one strand and discontinuous on the other.

Try it first, then show the solutionSolution

(a) Separating the two strands along the entire length of a long DNA would need a very high amount of energy, so it opens in a small region, the replication fork (1 mark).
(b) DNA polymerase can add nucleotides only in the 5'→3' direction (1 mark).
On the template with polarity 3'→5' the new strand grows continuously towards the fork; on the template with polarity 5'→3' it is made in short fragments away from the fork, later joined by DNA ligase (1 mark).

Answer: Energy constraint → fork; 5'→3' rule → continuous on 3'→5' template, discontinuous (ligase-joined) on 5'→3' template.

Example 3: Meselson and Stahl · 5 marks · Long answerHarder board level

Describe the Meselson and Stahl experiment and explain how its results support semiconservative replication. What would be the proportion of hybrid and light DNA after 80 minutes?

Try it first, then show the solutionSolution

E. coli was grown for many generations in ¹⁵NH₄Cl so its DNA became heavy, then transferred to ¹⁴NH₄Cl (1 mark).
DNA from samples taken at intervals was separated by CsCl density gradient centrifugation, which separates heavy, hybrid and light DNA (1 mark).
After one generation (20 min) all DNA was hybrid; after two generations (40 min) there were equal amounts of hybrid and light DNA (1 mark).
Only semiconservative replication gives these bands: each daughter molecule keeps one heavy parental strand and adds one light strand (1 mark).
80 min = 4 generations: 16 molecules, 2 hybrid and 14 light, so hybrid : light = 1 : 7 (1/8 hybrid) (1 mark).

Answer: Heavy → hybrid → 1 hybrid : 1 light; after 80 min, 1/8 hybrid and 7/8 light.

Example 4: Writing the new strand · 2 marks · Very short answerHarder board level

One strand of a DNA segment reads 3'-TACCGATTG-5'. Write the new strand formed on it during replication with its polarity. Will this new strand be made continuously or discontinuously if the fork moves in the direction in which this template is read 3'→5'?

Try it first, then show the solutionSolution

Complementary and antiparallel: 5'-ATGGCTAAC-3' (1 mark).
The template has polarity 3'→5' in the direction of fork movement, so the new strand grows 5'→3' towards the fork: continuously (1 mark).

Answer: 5'-ATGGCTAAC-3'; continuous (leading) strand.

Example 5: 60-minute sample · 2 marks · Very short answerHarder board level

E. coli grown in ¹⁵N medium is shifted to ¹⁴N medium and its DNA is extracted after 60 minutes. What proportion of the DNA is light and what proportion hybrid? Would any heavy DNA be seen?

Try it first, then show the solutionSolution

60 min = 3 generations: $2^{3}$ = 8 molecules, of which 2 are hybrid and 6 light (1 mark).
Hybrid 1/4 (25%), light 3/4 (75%); no heavy DNA, because both heavy strands are now in separate hybrid molecules (1 mark).

Answer: 25% hybrid, 75% light, no heavy.

Example 6: Testing the alternative model · 3 marks · Short answerHOTS / competency

Suppose replication were conservative (the parental double helix stays intact and a wholly new molecule is made). Predict the CsCl bands after one and two generations, and explain how the actual results rule this out.

Try it first, then show the solutionSolution

Conservative, generation 1: one heavy band (original molecule) and one light band (new molecule), in equal amounts (1 mark).
Generation 2: one heavy and three light (1 heavy : 3 light); no hybrid band ever (1 mark).
The actual result after one generation was a single hybrid band with no heavy band, which the conservative model cannot produce; so replication is semiconservative (1 mark).

Answer: Conservative: heavy + light (no hybrid); observed single hybrid band rules it out.

Where students lose marks in DNA Replication

The slips examiners see most often, and what to write instead.

Mistake: Saying the continuous strand forms on the 5'→3' template
Template and new-strand polarities are confused.
Wrong working
Replication is continuous on the template strand with 5'→3' polarity.
Right working
Continuous on the template with 3'→5' polarity; discontinuous on the 5'→3' template.
Do this: The new strand runs 5'→3', antiparallel to its template, so the template must be 3'→5'.
Usually costs: 1 mark.
Mistake: Expecting a heavy band after one generation
Students mix up semiconservative and conservative models.
Wrong working
After one generation: one heavy and one light band.
Right working
After one generation: one hybrid band only.
Do this: Every daughter molecule has one ¹⁵N and one ¹⁴N strand.
Usually costs: 1-2 marks.
Mistake: Wrong hybrid count
Students halve the hybrid amount each generation.
Wrong working
After 3 generations: 1 hybrid out of 8.
Right working
After 3 generations: 2 hybrid out of 8 (1/4).
Do this: Two old strands → always 2 hybrid molecules.
Usually costs: 1 mark.
Mistake: Forgetting the CsCl technique
Students describe only the isotopes.
Wrong working
They used ¹⁵N and ¹⁴N and found the DNA was semiconservative.
Right working
DNA was separated by density in a caesium chloride gradient by centrifugation.
Do this: Name organism, isotopes, technique, results, conclusion.
Usually costs: 1 mark in 5-mark answers.

Exam tips

Write isotopes as ¹⁵NH₄Cl and ¹⁴NH₄Cl, and mention that ¹⁵N is the heavy isotope.
Always mark 5' and 3' ends in the replication fork diagram; unmarked polarity loses marks.
Quote E. coli's 20-minute generation time when converting time to generations.
Give both reasons for the fork in one line: energy cost of separating the whole molecule.
Use the term 'DNA-dependent DNA polymerase' in full at least once.

Practice questions on DNA Replication

Write each answer on paper first. Open the hint only if you are stuck, and the answer only when you have finished.

Q1 · 1 mark · MCQBoard level
In the Meselson-Stahl experiment, DNA extracted one generation after transfer from ¹⁵N to ¹⁴N medium was:
(a) all heavy (b) all light (c) all hybrid (d) half heavy, half light
Hint
One old strand and one new.
Answer
(c) all hybrid.
Q2 · 1 mark · Assertion-reasonHarder board level
Assertion (A): DNA replication takes place in the S phase of the cell cycle.
Reason (R): DNA replication and cell division must be highly coordinated; failure of division after replication leads to polyploidy.
(a) Both A and R are true and R is the correct explanation of A.
(b) Both A and R are true but R is not the correct explanation of A.
(c) A is true but R is false.
(d) A is false but R is true.
Hint
Does R explain why it is the S phase?
Answer
(b). Both are true, but R explains the need for coordination, not why replication happens in S phase specifically.
Q3 · 1 mark · Very short answerBoard level
Name the enzyme that joins the discontinuously synthesised fragments, and the enzyme that polymerises deoxynucleotides.
Hint
Ligase and polymerase.
Answer
DNA ligase joins the fragments; DNA-dependent DNA polymerase polymerises deoxynucleotides.
Q4 · 2 marks · Very short answerHarder board level
Why must a piece of DNA be inserted into a vector to be copied in a bacterium? Link your answer to how replication starts.
Hint
Origin of replication.
Answer
Replication does not start at random; it begins only at a specific origin of replication, and DNA polymerase cannot initiate on its own. A foreign DNA piece without an ori would not be replicated, so it is linked to a vector that provides an origin.
Q5 · 4 marks · Case-basedHOTS / competency
Read and answer.
Suppose a researcher grows bacteria in ¹⁵N medium, transfers them to ¹⁴N medium and centrifuges DNA samples in CsCl. Tube A shows one band in the middle; Tube B shows two bands of equal intensity, middle and upper; Tube C shows two bands, middle and upper, with the upper band three times as intense.
(a) Arrange the tubes in order of time after transfer. (1)
(b) How many generations had passed for Tube C? (1)
(c) If the bacteria divide every 20 minutes, after how many minutes would the middle band be only 1/16 of the DNA? Show working. (2)
Hint
Hybrid fraction = 2/2^n.
Answer
(a) A (1 generation), B (2), C (3). (b) 3 generations (2 hybrid : 6 light = 1 : 3). (c) 2/2^n = 1/16 → 2^n = 32 → n = 5 generations = 5 × 20 = 100 minutes.
Q6 · 3 marks · Short answerHarder board level
Draw a labelled replication fork showing polarity. Explain why one new strand is synthesised in fragments.
Hint
Mark 5' and 3' on every strand.
Answer
Diagram: Y-shaped fork; template strands labelled 3'→5' and 5'→3'; a continuous new strand on the 3'→5' template growing towards the fork; short fragments on the 5'→3' template; DNA polymerase and DNA ligase labelled. Reason: DNA polymerase works only 5'→3'; on the 5'→3' template the new strand must grow away from the fork, so it is made in short pieces as the fork opens, later joined by DNA ligase.

Test your recall

Answer in your head or on paper first, then open the card. Pulling an answer out of memory makes it stick far better than re-reading.

1.Who proved semiconservative replication?Show answer
Meselson and Stahl (1958).
2.Which isotope made the DNA heavy?Show answer
¹⁵N (from ¹⁵NH₄Cl).
3.How was heavy DNA separated from light DNA?Show answer
CsCl density gradient centrifugation.
4.In which direction does DNA polymerase work?Show answer
5'→3'.
5.Fraction of hybrid DNA after 4 generations?Show answer
1/8.
6.Which enzyme joins discontinuous fragments?Show answer
DNA ligase.

DNA Replication: frequently asked questions

What is semiconservative replication?

Replication in which each daughter DNA molecule has one strand from the parent and one newly made strand.

What did the Meselson and Stahl experiment show?

After one generation in ¹⁴N, all DNA from ¹⁵N-labelled E. coli was hybrid; after two, half was hybrid and half light. This proved replication is semiconservative.

Why is one strand made discontinuously?

DNA polymerase adds nucleotides only in the 5'→3' direction, so on the 5'→3' template the new strand is made in short fragments that DNA ligase joins.

Why does replication happen at a fork?

Separating the two strands along the whole length of a long DNA would need too much energy, so the helix opens in a small region at a time.

What is the origin of replication?

The specific region on DNA where replication begins. DNA polymerase cannot start replication on its own, so a DNA piece needs an origin (provided by a vector) to be replicated.