Home › Learning Hub › IB DP Biology › D1.1 DNA replication
D1.1

DNA replication

Theme D · Continuity and change · Molecules · SL and HL · plus additional higher level

Every time a cell divides, its entire genome — about six billion bases in a human cell — must be copied, and copied almost perfectly. This topic explains how the structure of DNA makes that possible, and how biotechnology has borrowed the same machinery to copy DNA in a test tube. At HL, the topic looks closely at direction: because DNA polymerase can only build in one direction, the two strands have to be copied in different ways.

🎯What you need to be able to do

  • Explain why DNA replication is needed and why it must produce exact copies.
  • Explain semi-conservative replication and how complementary base pairing makes it accurate.
  • Outline the roles of helicase and DNA polymerase.
  • Explain how PCR amplifies DNA (primers, temperature changes, Taq polymerase) and how gel electrophoresis separates fragments.
  • Outline applications of PCR and gel electrophoresis, including DNA profiling for paternity and forensics.
  • AHL Explain the directionality of DNA polymerase and the difference between leading and lagging strands.
  • AHL Outline the roles of DNA primase, DNA polymerase I and III and DNA ligase in prokaryotes, and DNA proofreading.

📚The biology

Why DNA is replicated

DNA replication produces exact copies of a DNA molecule, with identical base sequences. It must happen before a cell divides, so that each daughter cell receives a complete set of genetic information. It is therefore needed for:

  • reproduction — asexual reproduction, and producing gametes in sexual reproduction;
  • growth of multicellular organisms, which grow by increasing their number of cells;
  • tissue replacement — replacing cells that die or are damaged, such as skin and blood cells.

Semi-conservative replication

DNA replication is semi-conservative: each new DNA molecule consists of one strand from the original molecule and one newly synthesized strand.

  1. The two strands of the double helix separate.
  2. Each strand acts as a template for a new strand.
  3. Free nucleotides are added to the new strand by complementary base pairing: A pairs with T, and G pairs with C.
  4. The result is two DNA molecules, each identical to the original, each containing one old and one new strand.

Complementary base pairing is what makes replication accurate: each base on the template can pair with only one type of base, so the sequence of the new strand is dictated exactly by the template. Only the correct nucleotide forms stable hydrogen bonds in the right positions.

Helicase and DNA polymerase

Helicase
Unwinds the double helix and breaks the hydrogen bonds between complementary bases, separating the two strands. It uses energy from ATP. The point where the strands separate is the replication fork.
DNA polymerase
Adds nucleotides one at a time to the new strand, matching each to the template by complementary base pairing, and links them into a sugar–phosphate backbone with covalent bonds.

The polymerase chain reaction (PCR)

PCR is a technique for amplifying DNA: making millions or billions of copies of a specific sequence from a tiny starting sample. A reaction tube contains the sample DNA, free nucleotides, primers and Taq polymerase, and a thermal cycler repeatedly changes the temperature:

1. Denaturation — about 95 °C
The high temperature breaks the hydrogen bonds between the strands, separating the DNA into single strands. (Helicase is not needed.)
2. Annealing — about 55–60 °C
On cooling, primers — short single-stranded DNA sequences complementary to the two ends of the target region — bind to the separated strands by base pairing. They mark where copying starts, and so define which sequence is amplified.
3. Extension — about 72 °C
Taq polymerase adds nucleotides to the primers, building complementary strands.

Taq polymerase is a DNA polymerase from Thermus aquaticus, a bacterium from hot springs. It is heat-stable: it is not denatured at 95 °C, so it survives repeated cycles and does not need to be added again each time. Its optimum is around 72 °C.

Each cycle takes a few minutes and doubles the amount of the target DNA. After \( n \) cycles, one molecule becomes \( 2^{n} \).

Gel electrophoresis

Gel electrophoresis separates DNA fragments by size:

  1. DNA samples are placed in wells at one end of an agarose gel, submerged in a buffer solution.
  2. An electric current is applied. DNA is negatively charged (because of its phosphate groups), so fragments move towards the positive electrode.
  3. The gel is a mesh. Smaller fragments move through it more easily and travel further in a given time; larger fragments travel less far.
  4. The DNA is stained so the fragments show up as bands. A ladder of fragments of known sizes is run alongside for comparison.

Applications of PCR and gel electrophoresis

The two techniques are used together in a broad range of applications: diagnosing infections (for example testing for viral RNA in COVID-19 tests), detecting genetic diseases, identifying species from environmental DNA (A3.1), studying evolutionary relationships, and above all DNA profiling.

DNA profiling compares regions of DNA that are highly variable between individuals, usually short tandem repeats (STRs): short sequences repeated a different number of times in different people. These regions are amplified by PCR and separated by electrophoresis, giving a pattern of bands (a profile) that is almost unique to each person.

Forensic investigations
DNA from a crime scene (blood, hair, skin cells) is profiled and compared with profiles from suspects. A match links a suspect to the scene; a mismatch can exclude them.
Paternity testing
A child inherits half its DNA from each parent, so every band in the child’s profile must be present in either the mother’s or the father’s. Bands not from the mother must come from the biological father.

Reliability increases with the number of markers. Two unrelated people might share the same profile at one STR by chance, but it is extremely unlikely that they share it at many. Using more markers (current forensic systems use around 20) reduces the probability of a false match to a tiny fraction.

Directionality of DNA polymerase AHL

Each strand of DNA has a 5′ end (a phosphate on carbon 5′ of the terminal sugar) and a 3′ end (an –OH on carbon 3′) (A1.2). DNA polymerases can only add nucleotides to the 3′ end of a growing strand: the 5′ phosphate of the new nucleotide is linked to the 3′ –OH of the strand. New DNA is therefore always built in the 5′ → 3′ direction. DNA polymerase also cannot start a new strand from nothing; it needs an existing 3′ end to add to.

Leading and lagging strands AHL

Because the two template strands are antiparallel, and new strands can only grow 5′ → 3′, the two new strands at a replication fork are made differently:

Leading strand
Synthesized continuously, in the same direction as the fork moves. It needs to be initiated with an RNA primer only once.
Lagging strand
Synthesized discontinuously, in short sections called Okazaki fragments, in the direction away from the fork. As the fork opens more template, a new fragment must be started, so an RNA primer is needed repeatedly, once for each fragment.

The enzymes of replication in prokaryotes AHL

Helicase
unwinds the helix and separates the strands at the replication fork.
DNA primase
synthesizes short RNA primers complementary to the template, providing a 3′ end for DNA polymerase to add to — once on the leading strand, and at the start of each Okazaki fragment.
DNA polymerase III
the main replicating enzyme: adds DNA nucleotides to the 3′ end of the primer, building the new strand 5′ → 3′. Also proofreads.
DNA polymerase I
removes the RNA primers and replaces them with DNA nucleotides.
DNA ligase
joins the Okazaki fragments by forming the final covalent bond in the sugar–phosphate backbone between adjacent sections, making a continuous strand.

Proofreading AHL

Occasionally a nucleotide with the wrong base is added. DNA polymerase III checks each new base pair as it goes. If the nucleotide at the 3′ terminal of the new strand is mismatched with the template, the enzyme removes it, moving back one position, and then replaces it with the correctly matched nucleotide before continuing. This proofreading reduces the error rate dramatically, to around one mistake per billion bases.

✏️Worked example

(a) A forensic sample contains a single copy of a target DNA sequence. Calculate the number of copies after 25 cycles of PCR, assuming each cycle doubles the DNA.
(b) Explain why the reaction is heated to 95 °C and then cooled to about 55 °C in each cycle.
(c) A child’s DNA profile has bands at 3.1, 4.5, 6.0 and 8.2 kb. The mother has bands at 3.1, 5.0, 6.0 and 7.4 kb. Man X has bands at 4.5, 6.8, 8.2 and 9.0 kb; man Y has bands at 2.2, 4.5, 5.0 and 7.4 kb. Deduce which man could be the father.
(d) AHL Explain why primers must be added repeatedly on the lagging strand.

(a)

\[ 2^{25} = 33\,554\,432 \approx 3.4 \times 10^{7}\ \text{copies} \]

(b) At 95 °C the hydrogen bonds between complementary bases break, separating the strands so each can act as a template. Cooling to about 55 °C allows the primers to bind (anneal) to their complementary sequences on the single strands; at 95 °C they would not stay attached.

(c) Every band in the child’s profile must come from one parent. The mother accounts for 3.1 and 6.0 kb. The remaining bands, 4.5 and 8.2 kb, must come from the father. Man X has both 4.5 and 8.2 kb, so he could be the father. Man Y has 4.5 but not 8.2 kb, so he is excluded.

(d) DNA polymerase can only add nucleotides to an existing 3′ end and only builds 5′ → 3′. On the lagging strand, that direction is away from the replication fork, so the strand can only be made in short Okazaki fragments. Each time the fork opens up more template, synthesis of a new fragment must begin, and each new fragment needs its own RNA primer laid down by DNA primase to provide a 3′ end.

Check it. 210 = 1024 ≈ 103, so 220 ≈ 106 and 225 = 220 × 32 ≈ 3 × 107, consistent with the exact answer. In (c), a quick check is that the child should share about half its bands with each parent, which man X does and man Y does not.
“A match proves guilt” or “man X is the father”. A profile can exclude someone with certainty, but a match only shows they could be the source or father; with few markers another person could match by chance. That is why many markers are used, and why the conclusion should be phrased as a probability.

📝Practise

Work through these on paper, then reveal the answer. Questions 5 and 6 are AHL.

1. Explain what is meant by semi-conservative replication.
In semi-conservative replication, the two strands of the parent DNA molecule separate and each acts as a template for a new complementary strand. Each of the two resulting DNA molecules therefore contains one original (conserved) strand and one newly synthesized strand. Because of complementary base pairing, both new molecules have base sequences identical to the original.
2. State the roles of helicase and DNA polymerase in DNA replication.
Helicase unwinds the double helix and breaks the hydrogen bonds between complementary base pairs, separating the two strands. DNA polymerase adds free nucleotides to the new strand, pairing each with the complementary base on the template strand, and links them to form the sugar–phosphate backbone.
3. Explain why Taq polymerase is used in PCR rather than DNA polymerase from human cells.
Each PCR cycle includes heating to about 95 °C to separate the DNA strands. Human DNA polymerase would be denatured at this temperature and would have to be added again every cycle. Taq polymerase comes from Thermus aquaticus, a bacterium living in hot springs, and is heat-stable: it is not denatured at 95 °C and has an optimum temperature of about 72 °C. It therefore survives all the cycles, allowing PCR to run automatically.
4. Explain how gel electrophoresis separates DNA fragments.
DNA fragments are loaded into wells in an agarose gel and an electric current is applied. DNA is negatively charged because of its phosphate groups, so it moves towards the positive electrode. The gel acts as a mesh: smaller fragments pass through more easily and move further in a given time than larger fragments. Fragments therefore separate into bands according to size, which can be estimated by comparison with a ladder of known sizes.
5. AHL Distinguish between replication of the leading strand and the lagging strand.
DNA polymerase III can only add nucleotides to the 3′ end, building 5′ → 3′. On the leading strand, this direction is towards the replication fork, so synthesis is continuous and an RNA primer is needed only once. On the lagging strand, this direction is away from the fork, so synthesis is discontinuous, in short Okazaki fragments, each needing its own RNA primer; the primers are later replaced with DNA by DNA polymerase I and the fragments are joined by DNA ligase.
6. AHL Outline the roles of DNA primase, DNA polymerase I and DNA ligase in prokaryotic DNA replication.
DNA primase synthesizes short RNA primers complementary to the template, giving DNA polymerase III a 3′ end to extend — once on the leading strand and at the start of every Okazaki fragment on the lagging strand. DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides. DNA ligase joins adjacent sections of new DNA (Okazaki fragments) by forming a covalent bond in the sugar–phosphate backbone, producing a continuous strand.

🔗Go deeper — other people’s work

These are external resources, not mine. If one stops working, tell me and everything above it on this page still stands.

  • DNA Learning Center (Cold Spring Harbor) — animations of DNA replication, PCR and gel electrophoresis.
  • Learn.Genetics (University of Utah) — virtual labs for PCR and gel electrophoresis.
  • HHMI BioInteractive — the DNA replication animation showing leading and lagging strands in real time.