D1.1 DNA replication

DNA replication copies genetic information through template strands, complementary pairing, enzyme action, proofreading, and laboratory amplification or separation techniques used to analyse DNA.

Learning objectives

D1.1.1DNA replication• Produces exact DNA copies before cell division• Maintains genetic continuity for reproduction, growth, and tissue replacement D1.1.2Semi-conservative replication• Each new DNA molecule has one original strand and one new strand• Complementary base pairing gives accurate copying; Meselson-Stahl isotope evidence supports the model D1.1.3Role of helicase and DNA polymerase• Helicase unwinds DNA and breaks hydrogen bonds between strands• DNA polymerase joins complementary nucleotides to build new strands D1.1.4PCR and gel electrophoresis• PCR amplifies selected DNA using primers, temperature cycles, and Taq polymerase• Gel electrophoresis separates DNA fragments by size and charge D1.1.5Applications• PCR and gel electrophoresis support DNA profiling• Applications include forensic identification and paternity testing D1.1.6(HL)—DNA polymerase directionality• DNA strands have 5' and 3' ends• DNA polymerase adds nucleotides to the 3' end, so new DNA forms 5' to 3'D1.1.7(HL)—Leading vs. lagging strand• Leading strand synthesis is continuous; lagging strand synthesis is discontinuous• Lagging strand forms Okazaki fragments using repeated RNA primers D1.1.8(HL)—Functions in replication• Prokaryotic model: primase, DNA polymerase III, DNA polymerase I, and ligase• Primase starts, polymerases extend/replace primers, and ligase joins fragments D1.1.9(HL)—DNA proofreading• DNA polymerase III removes mismatched nucleotides from the 3' end• Proofreading improves copying accuracy and reduces mutations

DNA replication produces a second DNA molecule with the same base sequence as the original, apart from rare errors.

chromosome DNA → replication during interphase → two DNA copies → cell division → one complete genome enters each daughter cell

A cell copies its DNA before dividing so each daughter cell receives a DNA copy.

Biological context	Why replication is required
reproduction	genetic information passes to new cells or offspring
growth	cell number increases without losing the genome
tissue replacement	new cells inherit the instructions needed for their function

Semi-conservative replication gives each daughter DNA molecule one parental strand and one newly synthesized strand.

Exposed template base	New nucleotide added
A	T
T	A
C	G
G	C

The sequence of each original strand determines a complementary new sequence. The two products therefore preserve the original base-pair sequence.

Semi-conservative means that each daughter double helix contains one parental strand and one newly synthesized strand—not that half of every strand is old.

E. coli were first grown with ¹⁵N so their DNA was heavy, then transferred to ¹⁴N medium. DNA from successive generations was separated by density-gradient centrifugation.

Sample	Observed DNA bands	What the result shows
before transfer	one heavy band	all DNA initially contains ¹⁵N
after one replication	one intermediate band	every molecule contains heavy and light material
after two replications	one intermediate + one light band	hybrid molecules persist while fully light molecules appear

The one-generation intermediate band rejects conservative replication, which predicts separate heavy and light bands. The two-generation pattern matches semi-conservative copying.

The isotopes label nitrogen in DNA; they do not make replication radioactive. Both ¹⁵N and ¹⁴N are stable isotopes.

Helicase unwinds the double helix and breaks the hydrogen bonds between complementary bases, exposing two template strands.

Free DNA nucleotides align with exposed bases by complementary base pairing.

DNA polymerase joins adjacent nucleotides by forming phosphodiester bonds in the sugar–phosphate backbone of each new strand.

Enzyme	Bonds affected	Job
helicase	hydrogen bonds between bases	separates the two strands
DNA polymerase	phosphodiester bonds within a new strand	links nucleotides into a backbone

Helicase does not elongate DNA. It opens the template; DNA polymerase performs strand synthesis.

need two genomes → helicase separates the original strands → each old strand acts as a template → complementary nucleotides pair → DNA polymerase links the new backbone → two matching double helices form

Feature to check	Correct outcome
sequence	complementary copying preserves the base-pair sequence
strand origin	each product has one old strand and one new strand
biological result	a complete genome can pass to each daughter cell

A model explains how copying could occur; the Meselson–Stahl band pattern provides experimental evidence that the one-old-one-new strand distribution actually occurs.

The polymerase chain reaction (PCR) amplifies a selected DNA region in vitro, producing enough copies for detection or analysis.

Component	Job
template DNA	contains the target sequence
two DNA primers	bind on opposite strands at the target's ends and provide free 3′ ends
free DNA nucleotides	become the new strands
Taq polymerase	extends primers and remains active after high-temperature heating

Primer sequences determine which interval is copied. The target's flanking sequences must therefore be known well enough to design complementary primers.

PCR combines template DNA, two primers, free nucleotides and Taq polymerase, then uses repeated temperature cycles to make many copies before gel separation.

Denaturation, about 95 °C: hydrogen bonds break and double-stranded DNA separates into templates.

Annealing, a lower temperature: primers bind to complementary sequences flanking the target.

Extension, about 72 °C: Taq polymerase adds nucleotides to each primer's 3′ end, synthesizing new DNA 5′→3′.

Heating begins the next cycle by separating the newly formed double strands. A thermal cycler repeats the temperature sequence automatically.

Cooling does not make DNA polymerase copy the target by itself. Cooling permits primer binding; polymerase extends only after a primer supplies a paired 3′ end.

$N_n=N_0(2^n)$

In the ideal early cycles, each target molecule produces two copies per cycle. Starting from one target, 30 perfect doublings would give about one billion copies.

Taq polymerase comes from the thermophilic bacterium Thermus aquaticus. Its heat stability prevents the enzyme from being destroyed during repeated denaturation.

Limitation	Consequence
primers bind a similar non-target sequence	the wrong interval can be amplified
contamination supplies foreign DNA	contaminant DNA can also be copied
reagents become limiting and products reanneal	later cycles fall below ideal doubling
Taq lacks proofreading	some newly copied molecules contain errors

DNA samples are loaded into wells near the negative electrode of a porous agarose gel immersed in conducting buffer.

Phosphate groups give DNA a net negative charge, so fragments migrate through the gel toward the positive electrode when voltage is applied.

Fragment	Movement through gel pores	Final tendency after the same time
shorter	less hindered; moves faster	farther from the wells
longer	more hindered; moves slower	nearer the wells

DNA is colourless. A stain or labelled probe is needed to reveal separated fragments as bands.

For DNA fragments, charge gives a common migration direction; fragment length is the main variable responsible for separation through the gel.

Observation	Interpretation
band farther from wells	shorter DNA fragments
bands at the same height in two lanes	fragments of the same measured length
more intense band	more stained DNA at that position, not a longer fragment

Compare lanes horizontally, one band position at a time. A profile comparison uses a pattern across many variable markers, not one matching band.

A same-height band supports a shared fragment length; it does not by itself prove that every base in the fragments is identical.

collect and preserve samples → extract DNA → amplify selected STR or VNTR markers by PCR → separate products by electrophoresis → compare band or peak patterns

Result	Supported conclusion	Not established by the profile alone
questioned sample differs at a reliable marker	that reference individual is excluded	who left the sample
all tested markers match	the samples may share a source	when or how the DNA arrived

Match strength increases when more independent, variable markers agree and when the matching pattern is rare in the relevant population.

Contamination, mixed samples, degraded DNA and laboratory error can weaken the inference. Controls and careful sample handling are part of the evidence.

At each tested marker, a child inherits one allele from the biological mother and one from the biological father.

Start with the child's pattern → account for bands or alleles shared with the known parent → compare every remaining child marker with the possible other parent.

Comparison	Inference
a required child marker is absent from the possible parent	exclude that person at that marker, after checking data quality
all non-maternal child markers are present in the possible father	parentage is supported, with strength depending on marker number and frequencies

Close relatives share more DNA than unrelated people. Parentage conclusions use multiple markers and probability, not a visual impression from one or two bands.

Stage	Question answered	Key control
primer design	which region will be copied?	complementary, target-specific flanking sequences
PCR	is there enough target DNA to analyse?	positive and negative controls; contamination prevention
electrophoresis	what fragment lengths are present?	ladder and correct electrode orientation
profile comparison	are patterns consistent with a shared source or inheritance?	enough informative markers and relevant frequencies

PCR changes quantity; electrophoresis changes position; interpretation changes neither. Each step supports a different claim.

A laboratory match is evidence of consistency. The final conclusion must remain limited by sample quality, contamination risk, marker informativeness and the biological question being tested.

The two DNA strands are antiparallel: one runs 5′→3′ while its complement runs 3′→5′.

DNA polymerase forms a phosphodiester bond between the growing strand's free 3′-OH and the incoming nucleotide. New DNA therefore grows only 5′→3′.

Polymerase action	Direction
reads template	3′→5′
synthesizes new strand	5′→3′

DNA polymerase reads a template from 3 prime to 5 prime while adding nucleotides to the new strand's 3 prime end, so the new strand grows 5 prime to 3 prime.

Both templates are exposed at the same fork, but DNA polymerase can extend each new strand only 5′→3′.

On one template, 5′→3′ synthesis follows the advancing fork, so polymerase can remain behind helicase and synthesize continuously.

On the opposite template, 5′→3′ synthesis points away from the fork. Newly exposed template must be copied in short sections that begin repeatedly near the fork.

The lagging strand is discontinuous because of antiparallel geometry and polymerase directionality—not because its polymerase is slower.

A replication fork shows continuous leading-strand synthesis and primer-dependent Okazaki fragments on the lagging strand.

Feature	Leading strand	Lagging strand
relation to fork movement	synthesis proceeds toward the fork	each fragment is synthesized away from the fork
synthesis pattern	continuous	discontinuous
RNA primers at one fork	one initial primer	repeated primers
DNA product before processing	one continuous new strand	multiple Okazaki fragments
ligase requirement	not required to join Okazaki fragments	joins processed fragments

Both new strands are synthesized simultaneously and both grow 5′→3′. The difference is continuous versus fragment-by-fragment synthesis.

The leading strand also needs an RNA primer to begin. Repeated priming is distinctive of the lagging strand.

DNA polymerase III cannot start a strand from unlinked nucleotides; it needs an existing paired chain with a free 3′-OH.

Primase is an RNA polymerase that synthesizes a short RNA primer complementary to the DNA template. It can start without a pre-existing 3′ end.

DNA polymerase III binds at the primer's 3′ end and adds complementary DNA nucleotides, extending the new strand 5′→3′.

New strand	Primase action	DNA polymerase III action
leading	makes one initial primer	extends continuously
lagging	makes a new primer for each fragment	extends each Okazaki fragment

DNA polymerase I removes each RNA primer and replaces its RNA nucleotides with DNA nucleotides.

After replacement, adjacent DNA sections contain the correct nucleotides but remain separated by a nick: one missing phosphodiester bond in the sugar–phosphate backbone.

DNA ligase catalyses formation of the missing phosphodiester bond, joining adjacent DNA fragments into one continuous strand.

Enzyme	Changes nucleotide identity?	Joins a final backbone gap?
DNA polymerase I	yes—RNA is replaced with DNA	leaves a nick between sections
DNA ligase	no	yes—seals the nick

Helicase: separates the parental strands at the replication fork.

Primase: makes an RNA primer that supplies a free 3′-OH.

DNA polymerase III: extends complementary DNA from the primer.

DNA polymerase I: removes the RNA primer and replaces it with DNA.

DNA ligase: seals the remaining nick between DNA sections.

Material present	Next enzyme needed
paired template but no starting chain	primase
RNA primer with free 3′ end	DNA polymerase III
RNA segment embedded before DNA	DNA polymerase I
adjacent DNA sections with a nick	DNA ligase

A newly added nucleotide that does not pair correctly distorts the polymerase–DNA structure and stalls extension.

DNA polymerase III detects a mismatched nucleotide at the new strand's 3 prime end, removes it and adds the correct complementary nucleotide.

DNA polymerase III shifts the 3′ end to its proofreading site, where exonuclease activity removes the mismatched nucleotide.

The corrected 3′ end returns to the polymerizing site; the complementary nucleotide is added and 5′→3′ synthesis resumes.

Proofreading greatly reduces replication errors and therefore mutation rate, but it cannot make replication completely error-free.

Clue at the fork	Consequence
polymerase can add only to a 3′ end	every new segment grows 5′→3′
synthesis can follow helicase	leading strand is continuous
synthesis must restart as template is exposed	lagging strand uses repeated primers and Okazaki fragments
an RNA primer remains	DNA polymerase I replaces it with DNA
two DNA sections meet at a nick	ligase seals the phosphodiester bond
the terminal base is mismatched	DNA polymerase III removes and replaces it

helicase opens → primase starts → DNA polymerase III extends → DNA polymerase I replaces RNA → ligase seals → polymerase III proofreading corrects many terminal mismatches

Directionality creates the leading–lagging difference. Enzyme handoffs solve that geometry, while complementary pairing plus proofreading preserves sequence fidelity.

8 marks

Growth in living organisms includes replication of DNA. Explain DNA replication.

3 marks

Outline the reason that DNA replication is described as semi-conservative.

1 mark

What is a function of the enzyme helicase?

4 marks

Describe the polymerase chain reaction (PCR).

4 marks

Outline the process of DNA profiling.

1 mark

How does DNA replicate?

1 mark

What is a difference between the leading and lagging strands in DNA replication?

3 marks

Describe the function of three named enzymes involved in DNA replication.

4 marks

Explain how mutation is avoided during DNA replication.

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D1.1 DNA replication

Learning objectives

DNA Must Be Copied Before a Cell Divides

Each Old DNA Strand Templates a New Complement

Meselson and Stahl Tested the Strand-Distribution Models

Helicase Exposes Templates; DNA Polymerase Builds Backbones

Reconstruct the Core Replication Argument

PCR Selects a DNA Region with Two Primers

One PCR Cycle Separates, Primes and Extends DNA

Repeated PCR Cycles Amplify—But Not Perfectly

An Electric Field Separates DNA Fragments through a Gel

Band Position Reports Fragment Length; Alignment Reports a Match

DNA Profiles Compare Several Variable Markers

A Child's Markers Must Be Accounted for by Both Parents

Follow DNA from a Tiny Sample to a Qualified Conclusion

DNA Polymerase Extends a Strand Only at Its 3′ End

Antiparallel Templates Force Two Replication Patterns

Leading Is Continuous; Lagging Uses Okazaki Fragments

Primase Starts; DNA Polymerase III Extends

DNA Polymerase I Replaces Primers; Ligase Seals Nicks

Replication Enzymes Hand the Growing DNA from One Job to the Next

DNA Polymerase III Proofreads the Growing 3′ End

Solve a Replication Fork from Direction to Fidelity

Next Topic

DNA replication

Semi-conservative replication

Role of helicase and DNA polymerase

PCR and gel electrophoresis

Applications exam focus

DNA polymerase directionality

Leading vs. lagging strand

Functions in replication

DNA proofreading