Why must DNA be replicated in living organisms?

So that each daughter cell produced by cell division receives a complete, identical copy of the genome. Cell division is needed for reproduction, and in multicellular organisms for growth and for replacing damaged or worn-out cells.

What does "semi-conservative" replication mean?

Each new DNA molecule is made of one strand from the original molecule (conserved) and one newly synthesised strand, built using the old strand as a template.

How does complementary base pairing make replication accurate?

Each base can only pair with one other (A with T, G with C), so the template fixes the sequence of the new strand. Each new strand is identical to the other parental strand, so both daughter molecules match the original.

Unwinds the double helix at the replication fork and separates the two strands by breaking the hydrogen bonds between complementary bases (using ATP). The sugar–phosphate backbones are not broken.

Role of DNA polymerase

Adds free nucleotides one at a time to form a new strand, choosing each by complementary base pairing with the template, and links them with covalent bonds into a sugar–phosphate backbone.

Meselson–Stahl — what was seen after one and after two generations in ¹⁴N?

One generation — a single band of intermediate (hybrid) density, ruling out conservative replication. Two generations — two equal bands, hybrid and light, ruling out dispersive replication. Only semi-conservative replication fits both.

The three temperature stages of a PCR cycle

Denaturation at about 95 °C (strands separate); primer annealing at about 55–65 °C (primers bind to each single strand); extension at about 72 °C (Taq polymerase builds new strands from the primers).

Why are primers needed in PCR, and why two of them?

DNA polymerase can only extend an existing strand, so a primer gives it a 3′ end to start from; the primers also select which sequence is copied. Two are needed, one complementary to each strand at either end of the target.

Why is Taq polymerase used in PCR?

It comes from Thermus aquaticus, a hot-spring bacterium, so it is heat-stable — it is not denatured at 95 °C — and works best at about 72 °C, the extension temperature.

How many copies of a target sequence does PCR give from one molecule after n cycles?

2ⁿ, because the number doubles every cycle. After 30 cycles that is about 1.07 × 10⁹ — over a billion.

What is the basis of separation in gel electrophoresis?

Size (length). DNA is negatively charged (phosphate groups), so it moves towards the positive electrode; smaller fragments pass more easily through the pores of the gel, so they travel farther in the same time.

How is a DNA profile used in a paternity case?

Every band in the child must come from the mother or the father. After removing the bands that match the mother, the remaining (paternal) bands must all be present in a man for him to be the possible father. One missing band excludes him.

Why do DNA profiles use many STR markers?

Unrelated people may share alleles at one locus by chance. The probability of sharing every one of many independent loci is the product of the individual probabilities, so more markers make a false match far less likely — reliability increases with the number of measurements.

In which direction do DNA polymerases build strands, and why?

5′→3′. They can only add the 5′ phosphate of a free nucleotide to the free 3′ –OH at the end of the growing strand.

Leading vs lagging strand

Leading — made continuously, towards the fork, from one RNA primer. Lagging — made discontinuously, away from the fork, as Okazaki fragments, each started from a new RNA primer.

Functions of DNA primase, DNA polymerase III, DNA polymerase I and DNA ligase

Primase makes RNA primers. Pol III extends primers by adding DNA nucleotides to the 3′ end (and proofreads). Pol I removes RNA primers and replaces them with DNA. Ligase seals the nicks between fragments by forming a sugar–phosphate bond.

What is DNA proofreading?

DNA polymerase III detects a mismatched base on the nucleotide it has just added, removes that nucleotide from the 3′ end of the new strand, and replaces it with a nucleotide carrying the correctly matched base.

D1.1 DNA replication – Interactive IB Biology

Replication makes exact copies of DNA

SL & HLSyllabus understandings: D1.1.1

DNA replication is the production of new DNA molecules with exactly the same base sequence as the original. A cell replicates all of its DNA before it divides, so that each daughter cell receives a complete copy of the genome. (In eukaryotes this happens during the S phase of interphase.)

Cell division, and therefore replication, is needed for:

Reproduction: unicellular organisms reproduce by cell division, and multicellular organisms use cell division to produce the cells that become gametes. Base sequences are passed from generation to generation.
Growth: a multicellular organism grows by producing more cells, each with the same genome.
Tissue replacement: cells lost through damage or wear, such as skin and gut lining cells, are replaced by division of other cells.

Because the copies are so accurate, the genetic information in every body cell is (almost) identical, and offspring inherit the same base sequences their parents carried. Replication is a process of continuity rather than change. Rare copying errors are one source of mutations (see D1.3 Mutation and gene editing).

The base sequence carries the information that is transcribed and translated into polypeptides (D1.2 Protein synthesis). Because copies must be faithful, replication has several safeguards built in: the two strands are used as templates, bases pair only in complementary pairs, and (higher level) DNA polymerase checks its own work.

Semi-conservative replication and complementary base pairing

SL & HLSyllabus understandings: D1.1.2

Replication begins by separating the two strands of the parent molecule. Each single strand is then used as a template for a new strand. Every new DNA molecule therefore consists of:

one strand conserved from the parent molecule, and
one newly synthesised strand.

This is semi-conservative replication (“half-conserving”).

Why the copies are identical

New strands are assembled by complementary base pairing: adenine pairs only with thymine and guanine only with cytosine. So the base sequence of each new strand is decided by its template:

Strand	Base sequence (written base for base under each other)
Parental strand 1	A C G T T G C
Parental strand 2	T G C A A C G
New strand on template 1	T G C A A C G (same as strand 2)
New strand on template 2	A C G T T G C (same as strand 1)

Each new strand is complementary to its template and identical to the other parental strand. Both daughter molecules therefore have the same base sequence as the original.

How this gives high accuracy

Only one base fits opposite each template base, so the chance of inserting the wrong one is very small.
A complete parental strand is kept in each molecule, so the original sequence is never lost: it is the reference against which the new strand is made (and, at higher level, checked).

Evidence: the Meselson–Stahl experiment

In 1958 Matthew Meselson and Franklin Stahl grew E. coli on the heavy isotope of nitrogen, ¹⁵N, then moved them to ordinary ¹⁴N and separated their DNA by density.

The Meselson–Stahl experiment

Generation 0 — all ¹⁵N. After many generations in ¹⁵N medium, all the bacterial DNA contains heavy nitrogen in both strands. Centrifuged in caesium chloride, it forms a single band low in the tube (heavy).

One generation in ¹⁴N. The bacteria are moved to ¹⁴N medium and allowed to divide once. All the DNA now forms a single band halfway between heavy and light — every molecule is a hybrid of one ¹⁵N strand and one ¹⁴N strand.

Two generations in ¹⁴N. After a second division there are two bands of equal size — one hybrid and one light (¹⁴N only). The original ¹⁵N strands are still intact, each now paired with a new ¹⁴N strand.

Which model fits?. Conservative replication predicts heavy and light bands after one generation, but there was only a hybrid band. Dispersive replication predicts a single band after two generations, but there were two. Only semi-conservative replication predicts both results.

Text description of this animation

Bacteria (E. coli) were grown for many generations in a medium whose only nitrogen source was the heavy isotope ¹⁵N, so all their DNA was heavy. They were then moved to a medium containing only ¹⁴N. DNA was extracted at intervals and spun at high speed in a caesium chloride solution, which forms a density gradient; each type of DNA settles as a band where its density matches the solution. Generation 0 gives one low (heavy) band. After one generation in ¹⁴N there is a single band of intermediate (hybrid) density. After two generations there are two bands of equal size, one hybrid and one light. The last step compares these results with the predictions of the conservative model (heavy and light bands after one generation, no hybrid) and the dispersive model (a single band that becomes lighter each generation): only semi-conservative replication fits.

Generation 0 — all ¹⁵N. After many generations in ¹⁵N medium, all the bacterial DNA contains heavy nitrogen in both strands. Centrifuged in caesium chloride, it forms a single band low in the tube (heavy).
One generation in ¹⁴N. The bacteria are moved to ¹⁴N medium and allowed to divide once. All the DNA now forms a single band halfway between heavy and light — every molecule is a hybrid of one ¹⁵N strand and one ¹⁴N strand.
Two generations in ¹⁴N. After a second division there are two bands of equal size — one hybrid and one light (¹⁴N only). The original ¹⁵N strands are still intact, each now paired with a new ¹⁴N strand.
Which model fits?. Conservative replication predicts heavy and light bands after one generation, but there was only a hybrid band. Dispersive replication predicts a single band after two generations, but there were two. Only semi-conservative replication predicts both results.

The single hybrid band after one generation ruled out conservative replication; the two bands (hybrid and light) after two generations ruled out dispersive replication.

Helicase and DNA polymerase

SL & HLSyllabus understandings: D1.1.3

The replication fork in 3D

Parental strands are dark and new DNA is red, so each daughter molecule shows one of each.

Text description of this visual

A DNA molecule 24 base pairs long lies across the screen. On the right it is still a right-handed double helix of two antiparallel strands; the parental strands have dark backbones and bases coloured as in A1.2 (adenine blue, thymine orange, guanine green, cytosine purple), with two hydrogen bonds (dashed yellow) in each A–T pair and three in each G–C pair. A purple ring of helicase moves from left to right along the molecule, unwinding the helix and breaking the hydrogen bonds, so the two strands separate into the two arms of a Y-shaped replication fork. Free nucleotides gather, and DNA polymerase (blue) links nucleotides with bases complementary to each template strand into a new strand with a red backbone. When the whole molecule has been copied there are two double helices, each with one dark parental strand and one red new strand: replication is semi-conservative. A second model (higher level) shows the prokaryotic fork in detail — RNA primers (amber, with uracil) laid by DNA primase, the leading strand made continuously towards the fork, the lagging strand made away from the fork as Okazaki fragments, DNA polymerase I replacing primers with DNA, DNA ligase sealing the nick, and DNA polymerase III proofreading a mismatched base.

Replication fork

Helicase: A ring-shaped enzyme that moves along the DNA at the fork. It unwinds the double helix and breaks the hydrogen bonds between complementary bases, separating the two strands. It uses energy from ATP.
Replication fork: The Y-shaped region where the parental double helix is being separated into two single strands. It moves along the molecule as replication proceeds.
Parental double helix: The part of the original molecule not yet copied. Its two strands are antiparallel and held together by hydrogen bonds between complementary bases.
Hydrogen bonds: Dashed yellow sticks — two in each A–T pair and three in each G–C pair. They are weak individually, so helicase can break them without breaking the covalent bonds of the sugar–phosphate backbones.
Template strand: Each separated parental strand is a template. Its exposed bases determine the order of bases in the new strand, because each base can only pair with its complementary base.
Free nucleotides: Nucleotides carrying all four bases are present in the nucleoplasm (or cytoplasm in prokaryotes). They are the raw material for the new strands.
DNA polymerase: Moves along a template strand, adding nucleotides whose bases are complementary to the template and linking each one to the end of the new strand by a covalent bond in the sugar–phosphate backbone.
New strand: Built from free nucleotides (red backbone). Its base sequence is complementary to its template and identical to the other parental strand.
Conserved parental strand: Each daughter molecule keeps one complete strand from the parent molecule (dark backbone). It was not broken up or copied — it acted as the template.
Two daughter DNA molecules: Both daughter molecules have exactly the same base sequence as the original molecule and as each other. Each is one parental and one new strand.
A–T pair: Adenine (blue) pairs only with thymine (orange), held by two hydrogen bonds.
G–C pair: Guanine (green) pairs only with cytosine (purple), held by three hydrogen bonds.

Helicase unwinds the helix. Helicase moves along the DNA, unwinding the double helix and breaking the hydrogen bonds between the complementary bases. The two strands separate, forming a Y-shaped replication fork. The covalent sugar–phosphate backbones are not broken.
Each strand is a template. The bases on each separated strand are now exposed. Each parental strand acts as a template for a new strand. Free nucleotides with all four bases are available nearby.
DNA polymerase builds new strands. DNA polymerase adds nucleotides one at a time, choosing each by complementary base pairing (A with T, G with C), and joins it to the new strand with a covalent bond. The two new strands are built in opposite directions because the templates are antiparallel.
Two identical daughter molecules. When the whole molecule has been copied, each daughter DNA molecule consists of one parental strand (dark) and one new strand (red), which wind back into a double helix. This is semi-conservative replication. Both molecules have the same base sequence as the original. (Simplified — the enzymes are not shown and both new strands are drawn as continuous; the AHL model shows the lagging strand.)

Prokaryotic fork (AHL)

Helicase: Unwinds the double helix and breaks the hydrogen bonds at the fork. In bacteria it is a ring of six subunits that encircles one template strand and moves along it towards the fork.
Direction of the fork: The arrow shows the fork moving to the right. The upper template runs 5′→3′ towards the fork; the lower template runs 3′→5′ towards the fork.
DNA primase: Makes an RNA primer — a short length of RNA nucleotides — by complementary base pairing with the template. DNA polymerase III cannot start a new strand without one.
RNA primer: A short RNA sequence (amber backbone; uracil instead of thymine) with a free 3′ end to which DNA polymerase III can add DNA nucleotides. The leading strand needs only this one primer.
DNA polymerase III (leading strand): Adds DNA nucleotides to the 3′ end of the primer and then of the growing strand, moving in the same direction as the fork, so the leading strand is made continuously.
DNA polymerase III (lagging strand): Extends each lagging-strand primer 5′→3′, which means moving away from the fork, until it reaches the previous fragment. Then it starts again at a new primer near the fork.
Leading strand: The new strand whose 5′→3′ direction is towards the fork. It is synthesised continuously and needs only one RNA primer.
3′ end of the leading strand: New nucleotides are only ever added here. The phosphate on carbon 5′ of the incoming nucleotide is bonded to the –OH on carbon 3′ of the last nucleotide in the strand.
Lagging strand: The new strand whose 5′→3′ direction is away from the fork. It is synthesised discontinuously, as a series of Okazaki fragments, each needing its own primer.
Okazaki fragment: A short section of lagging strand made from one RNA primer (about 1000–2000 nucleotides in bacteria; much shorter here). Each fragment is started close to the fork and extended away from it.
DNA polymerase I: Removes the RNA nucleotides of a primer and replaces them with DNA nucleotides, working from the 3′ end of the next fragment. It cannot join the last nucleotide to the strand ahead, so it leaves a nick.
Nick: A break in the sugar–phosphate backbone between two adjacent nucleotides on the lagging strand — a missing covalent bond between a 3′–OH and the 5′ phosphate next to it.
DNA ligase: Seals each nick by forming the missing covalent (phosphodiester) bond in the sugar–phosphate backbone, joining the Okazaki fragments into one continuous strand.
Mismatched nucleotide: A nucleotide with guanine was added opposite thymine. It does not pair properly, so DNA polymerase III removes it from the 3′ end before adding the correct nucleotide, with adenine.

Primase lays RNA primers. Helicase opens the fork. DNA primase makes a short RNA primer on each template, by complementary base pairing (uracil pairs with adenine). DNA polymerase III can only add nucleotides to an existing 3′ end, so it cannot start a strand without a primer.
Leading strand — continuous. DNA polymerase III adds DNA nucleotides to the 3′ end of the leading-strand primer. Because this strand is made 5′→3′ towards the fork, the polymerase can follow the fork and synthesise it continuously. On the other template, Pol III extends the first lagging-strand primer away from the fork.
Lagging strand — Okazaki fragments. As the fork opens more of the upper template, primase lays a new primer close to the fork. DNA polymerase III extends it away from the fork until it reaches the previous fragment. The lagging strand is made discontinuously, as Okazaki fragments, each with its own primer.
DNA polymerase I replaces primers. DNA polymerase I removes the RNA nucleotides of the earlier primer one by one and replaces them with DNA nucleotides, extending the 3′ end of the newer fragment. It cannot join its last nucleotide to the DNA ahead, so a nick is left in the backbone.
DNA ligase seals the nick. DNA ligase forms the missing covalent bond between the sugar of one nucleotide and the phosphate of the next, so the Okazaki fragments are joined into one continuous DNA strand.
Proofreading by DNA polymerase III. DNA polymerase III adds a nucleotide with guanine opposite thymine — a mismatch. It detects the mispairing, removes the incorrect nucleotide from the 3′ end of the new strand, and then adds a nucleotide with the correct base (adenine). This greatly reduces errors in replication.

Replication is carried out by a team of enzymes working together at a replication fork, the Y-shaped region where the parent molecule is being separated. The fork moves along the molecule as copying proceeds. At standard level you need the roles of two enzymes.

Helicase

Moves along the DNA at the fork.
Unwinds the double helix.
Breaks the hydrogen bonds between complementary bases, separating the two strands.
Uses energy from ATP.

Only hydrogen bonds are broken: the covalent bonds of each sugar–phosphate backbone stay intact, so each strand remains a continuous template.

DNA polymerase

Moves along each template strand.
Adds free nucleotides one at a time, each with the base that is complementary to the next base on the template.
Links each new nucleotide to the end of the growing strand by a covalent bond between the sugar of one nucleotide and the phosphate of the next, forming the new sugar–phosphate backbone.

After synthesis, each template strand and its new partner wind together into a double helix, giving two daughter DNA molecules.

PCR and gel electrophoresis

SL & HLSyllabus understandings: D1.1.4

The polymerase chain reaction (PCR)

PCR amplifies a chosen DNA sequence, turning a tiny sample into millions or billions of copies. The tube contains:

the DNA sample (the template);
primers: two short, single-stranded DNA sequences complementary to the two ends of the target sequence, one for each strand;
free nucleotides with all four bases;
Taq polymerase, a DNA polymerase from Thermus aquaticus, a bacterium that lives in hot springs. It is heat-stable, so it is not denatured at 95 °C.

A thermocycler repeats a cycle of temperature changes, typically 25–35 times:

Stage	Temperature	What happens
Denaturation	about 95 °C	Hydrogen bonds break and the strands separate
Primer annealing	about 55–65 °C	Primers bind to complementary sequences on each single strand
Extension	about 72 °C	Taq polymerase extends each primer, building a complementary strand

Each cycle doubles the number of copies of the target: after n cycles, one molecule gives 2ⁿ copies (2³⁰ is over a billion).

Simulation The polymerase chain reaction

Cycles completed: 0
Stage: —
Temperature: 25 °C
Copies of the target: 1

Jump to cycles completed: 0Starting copies of the target: 1Efficiency (share of molecules copied per cycle): 100%

Denaturation, about 95 °C. Heat breaks the hydrogen bonds between the bases, so the two strands of each DNA molecule separate.
Primer annealing, about 60 °C. Cooling lets the two primers form hydrogen bonds with complementary sequences at the 3′ ends of the target region, one on each single strand.
Extension, about 72 °C. Taq polymerase, which is not denatured at high temperature, adds nucleotides to the 3′ end of each primer, building a new strand complementary to each template.

this run perfect doubling (shown when efficiency is below 100%)

Before the first cycle: 1 copy of the target sequence.

Show data table

Cycles	Copies
0	1
5	32
10	1,024
15	32,768
20	1.05 × 10⁶
25	3.36 × 10⁷
30	1.07 × 10⁹
35	3.44 × 10¹⁰

Simplified model. Each completed cycle copies a set share of the molecules (perfect PCR doubles them: start × 2ⁿ). Real reactions slow to a plateau after about 30 cycles as primers and nucleotides are used up. Temperatures are typical: the annealing temperature (about 50–65 °C) is chosen to suit the primers.

Text description of this simulation

A schematic of one DNA molecule in a PCR tube with a thermometer, and a counter. Pressing "Next stage" steps through a cycle: denaturation at about 95 °C separates the strands; primer annealing at about 60 °C lets two primers bind to the 3′ ends of the target sequence, one on each strand; extension at about 72 °C lets Taq polymerase build new strands from the primers. After each complete cycle the number of copies of the target doubles (start × 2ⁿ), shown on a graph with a logarithmic scale; after 30 cycles one molecule has become over a billion. Sliders set the number of cycles, the starting number of copies and the efficiency of each cycle.

Gel electrophoresis

Gel electrophoresis separates DNA fragments by size (length).

Samples are loaded into wells at one end of an agarose gel, a mesh of polymer fibres in a buffer solution.
A voltage is applied. DNA is negatively charged because of its phosphate groups, so it moves towards the positive electrode. The wells are therefore placed at the negative end.
The gel mesh slows larger fragments more than smaller ones, so shorter fragments travel farther in the same time. Charge does not separate them, because it is proportional to length.
The current is switched off before the smallest fragments run off the end; the DNA is stained so the bands can be seen. Each band is many fragments of the same length.
A DNA ladder of fragments of known sizes is run in one lane, so the sizes of other bands can be estimated.

Applications: DNA profiling and more

SL & HLSyllabus understandings: D1.1.5

PCR and gel electrophoresis are used together in many fields. The most important example is DNA profiling.

DNA profiling

Human DNA contains short tandem repeats (STRs): places where a short sequence of a few bases is repeated many times. The number of repeats at each locus varies greatly between people, and each person has two versions (alleles), one from each parent.

DNA is extracted from a sample (blood, hair roots, skin cells, saliva or semen). Only a tiny amount is needed.
PCR amplifies a set of STR loci, using primers that bind either side of each repeat.
Gel electrophoresis separates the products by length: more repeats make a longer fragment, which moves less far. The pattern of bands is the DNA profile.

Simulation Gel electrophoresis and DNA profiling

Run time: 60 minMeasuring line (distance from the wells): 50

Move the measuring line onto a band to estimate its size from the ladder.

STR markers used in the profile: 13

Chance that an unrelated person matches at all 13 markers: about 1 in 10¹³.

After 60 minutes the fragments have separated by size. Ladder bands run from 3000 bp nearest the wells to 100 bp farthest away.

Check your conclusion

Suspect 2 has every band that is in the crime-scene sample, and no others, so their DNA profile matches. Suspects 1 and 3 share some bands with the sample but not all, so they are excluded as the source.

Qualitative model with invented profiles at three STR loci (two bands per person per locus). Distance travelled is taken to fall in proportion to log(fragment length), which is roughly true over the working range of an agarose gel. The match probability assumes each marker is shared by chance 1 time in 10 (illustrative).

Text description of this simulation

An agarose gel seen from above, with wells at the top next to the negative electrode. A DNA ladder of fragments of known size runs in the first lane next to four DNA profiles. Increasing the run time moves the negatively charged DNA towards the positive electrode, the smallest fragments farthest; if the gel runs too long the smallest fragments run off the end. A measuring line estimates the size of a band from the ladder. In the forensic case the crime-scene sample is compared with three suspects; in the paternity case a child is compared with the mother and two men. A slider shows how using more STR markers makes a chance match between unrelated people less likely.

Forensic investigations: a profile from a crime scene is compared with profiles from suspects (and victims). A match is strong evidence that the DNA came from that person; a mismatch at any locus excludes them.
Paternity cases: every band in a child’s profile must come from either the mother or the biological father. Ignore the child’s bands that match the mother; a man can be the father only if he has all the remaining bands.

Other applications

Diagnosing infections: tests for COVID-19 used RT-PCR, in which reverse transcriptase first copies the viral RNA into DNA, which PCR then amplifies; other pathogens are detected in the same way.
Genetic testing for alleles that cause disease, before birth or in adults.
Amplifying DNA for sequencing, for genetic modification, and from tiny or ancient samples in evolutionary studies.

DNA polymerases work in one direction

AHLSyllabus understandings: D1.1.6

Each strand of DNA has two different ends (see A1.2 Nucleic acids):

the 5′ end has a phosphate group attached to carbon 5′ of the last deoxyribose;
the 3′ end has a free –OH group on carbon 3′ of the last deoxyribose.

All the nucleotides in a strand point the same way, because every link in the backbone joins carbon 3′ of one sugar to the phosphate on carbon 5′ of the next.

The rule for DNA polymerases

DNA polymerases can only add nucleotides to the 3′ end of a strand. The phosphate on carbon 5′ of the incoming nucleotide is bonded to the –OH on carbon 3′ of the last nucleotide in the strand. So:

new strands always grow in the 5′→3′ direction;
because the new strand is antiparallel to its template, the polymerase moves along the template in the 3′→5′ direction;
the 5′ end of a strand is never extended.

Free nucleotides arrive as deoxynucleoside triphosphates. Two of the three phosphates are released as each nucleotide is added, which supplies the energy for the reaction.

A DNA polymerase also cannot start a new strand from nothing: it needs an existing 3′ end to add to. In the cell this is provided by an RNA primer.

Leading and lagging strands

AHLSyllabus understandings: D1.1.7

At a replication fork the two template strands are antiparallel, but both new strands must be built 5′→3′. The two strands are therefore made in different ways.

Leading strand: continuous

On one template, synthesis in the 5′→3′ direction runs towards the fork. DNA polymerase III can follow the fork as it opens, adding nucleotides without stopping. The leading strand is synthesised continuously, and an RNA primer is needed only once, at the start.

Lagging strand: discontinuous

On the other template, synthesis in the 5′→3′ direction runs away from the fork. As the fork opens, a new stretch of template is exposed behind the polymerase. Synthesis must keep restarting close to the fork with a new RNA primer, and each section is extended away from the fork until it meets the previous one. The lagging strand is therefore made discontinuously, as short sections called Okazaki fragments (about 1000–2000 nucleotides long in bacteria and 100–200 in eukaryotes).

	Leading strand	Lagging strand
Direction of synthesis relative to the fork	towards	away from
Synthesis	continuous	discontinuous (Okazaki fragments)
RNA primers needed	one	one per Okazaki fragment
Joining needed	no	yes, by DNA ligase

In bacteria replication starts at a single origin on the circular chromosome and two forks move away from it in opposite directions, each with its own leading and lagging strand.

Primase, DNA polymerase III, DNA polymerase I and ligase

AHLSyllabus understandings: D1.1.8

In prokaryotes, the main enzymes at the fork are:

Enzyme	Function
Helicase	Unwinds the double helix and breaks hydrogen bonds between bases at the fork
DNA primase	Makes RNA primers — short RNA sequences complementary to the template — giving a free 3′ end to build on: once on the leading strand, repeatedly on the lagging strand
DNA polymerase III	The main replicating enzyme: adds DNA nucleotides to the 3′ end of a primer and then of the growing strand, building the leading strand continuously and each Okazaki fragment
DNA polymerase I	Removes the RNA nucleotides of each primer and replaces them with DNA nucleotides, extending the 3′ end of the adjacent, newer (fork-side) Okazaki fragment; it leaves a nick
DNA ligase	Seals each nick by forming the missing covalent (sugar–phosphate) bond, joining Okazaki fragments into a continuous strand

Order of events on the lagging strand

Primase lays an RNA primer near the fork.
DNA polymerase III extends it away from the fork, making an Okazaki fragment, until it reaches the previous fragment’s primer.
DNA polymerase I replaces that primer with DNA.
DNA ligase seals the nick that remains.

Because the primers were made of RNA (with uracil and ribose), replacing them leaves the finished strand made entirely of DNA.

Proofreading by DNA polymerase III

AHLSyllabus understandings: D1.1.9

DNA polymerase III usually adds the correct nucleotide, but occasionally it inserts one whose base does not pair with the template, for example guanine opposite thymine. A mismatched base pair does not have the correct shape and hydrogen bonding.

Proofreading happens straight away:

DNA polymerase III detects that the base pair it has just made is mismatched.
It moves back and removes the incorrect nucleotide from the 3′ end of the new strand.
It then adds a nucleotide with the correctly matched base and carries on.

Proofreading reduces the error rate of replication by roughly a hundredfold, to about one wrong base in ten million added. Other repair systems correct many of the remaining errors, but you only need to know proofreading by DNA polymerase III. Replication errors that are not corrected become mutations that are passed on when the cell divides (see D1.3 Mutation and gene editing).

The last step of the AHL model shows proofreading at the 3′ end of the leading strand.

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D1.1 DNA replication

Big questions