DP Biology HL D1.1 DNA Replication

DP Biology HL D1.1 DNA Replication - Full notes

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DP Biology HL | D1.1 DNA Replication

DP BIOLOGY · THEME D: CONTINUITY AND CHANGE

D1.1

DNA Replication

Full notes for HL · IB-style

Contents

1	Why DNA must be replicated
2	Semi-conservative replication
3	The roles of helicase and DNA polymerase
4	Evidence for semi-conservative replication
5	The polymerase chain reaction (PCR)
6	Gel electrophoresis and its applications
7	Directionality: leading and lagging strands (HL only)
8	The enzymes of replication and proofreading (HL only)
9	Why DNA must be replicated
10	Semi-conservative replication
11	The Meselson–Stahl experiment
12	Helicase and unwinding
13	DNA polymerase and complementary pairing
14	Leading and lagging strands
15	Primers and primase
16	Proofreading and accuracy
17	The polymerase chain reaction (PCR)
18	Taq polymerase
19	Gel electrophoresis
20	DNA profiling
21	Mixed review: worked problems
22	Additional exam-style practice
23	Replication in prokaryotes and eukaryotes
24	Telomeres
25	Practical: modelling replication
26	Common misconceptions and links
27	Exam-style question with markscheme
28	Check your understanding
29	Summary and topic map

These are full teaching notes. Read each section in order, try the Think about it question before you read on, and do every Quick check before moving to the next section.

Before you start

Rate yourself on each statement now, and again after you've worked through the notes. Shade R (not yet), A (getting there) or G (confident).

I can…	R	A	G
explain why DNA replication is needed
explain semi-conservative replication and the role of complementary base pairing
describe the roles of helicase and DNA polymerase
outline the Meselson-Stahl evidence for semi-conservative replication
explain the stages of PCR and the role of Taq polymerase
explain gel electrophoresis and its use in DNA profiling
explain 5' to 3' synthesis, leading and lagging strands and Okazaki fragments
explain the roles of primase, DNA polymerase I and III, and DNA ligase
explain proofreading by DNA polymerase

Sections marked HL only (Sections 7 and 8) are Additional Higher Level content. Every other section is common to SL and HL.

Guiding questions

How is new DNA produced so accurately? How can scientists copy and compare DNA in the laboratory?

How this topic is examined

D1.1 is examined through diagrams of the replication fork, data from the Meselson-Stahl experiment, PCR stages and gel electrophoresis results (paternity and forensic profiles). HL questions focus on leading and lagging strands and the roles of each enzyme.

Use precise names for enzymes and say exactly what each one does.

Command term	What the examiner wants
State / Identify	a brief answer with no explanation needed
Outline	a brief account or summary
Describe	a detailed account, including trends or features, without reasons
Explain	a detailed account including causes, reasons or mechanisms
Distinguish / Compare and contrast	give the differences (and similarities), point by point
Calculate	a numerical answer with working and units
Deduce / Suggest	reach a conclusion from data; propose a reasonable explanation
Evaluate / Discuss	weigh strengths and limitations and reach a judgement

How to use these notes

Each section teaches one part of the syllabus in full. You'll see these boxes throughout; each has one job:

Box	What it's for
Think about it	a question to get you thinking before a section; the answer comes in the section
Key point	the one idea you must not forget
Common mistake	an error examiners see every year, and how to avoid it
In the real world	where the biology shows up outside the classroom
Try this	something quick you can do yourself to test the idea
Worked example	full solutions with IB-style marks (M1 method, A1 answer, R1 reasoning)
Quick check	short questions to test yourself at the end of each section

1 Why DNA must be replicated

Every new cell needs a complete copy of the genome.

Think about it

You began as one cell. Every one of your 30 trillion cells now contains a copy of the same DNA. How was it copied so many times without serious errors?

DNA replication is the production of exact copies of DNA. It is needed for reproduction (passing genetic information to offspring) and for growth and tissue replacement in multicellular organisms, because each new cell produced by cell division must receive a complete set of genetic information. Replication happens before cell division, during the S phase of the cell cycle (D2.1).

Replication must be highly accurate: errors (mutations) can harm or kill cells. In humans, the error rate after proofreading and repair is about one mistake in a billion bases copied.

2 Semi-conservative replication

Each new molecule keeps one old strand.

DNA replication is semi-conservative: each new DNA molecule consists of one original (parental) strand and one newly synthesized strand. The two strands of the double helix separate, and each acts as a template for a new complementary strand.

Accuracy depends on complementary base pairing: free nucleotides can only pair with the matching base on the template (A with T, C with G). So each new strand has exactly the base sequence complementary to its template, and the two daughter molecules are identical to the original.

Figure 1. Semi-conservative replication.

Key point

Semi-conservative: every daughter molecule is half old, half new. Complementary base pairing makes the copy accurate.

3 The roles of helicase and DNA polymerase

Unzip, then build.

Helicase unwinds the double helix and separates the two strands by breaking the hydrogen bonds between complementary bases. This creates a replication fork.

DNA polymerase moves along each template strand, adding free nucleotides (which are present in the nucleus as deoxynucleoside triphosphates) to the new strand, matching each base to its complementary partner on the template. It joins nucleotides to the growing strand by forming covalent (phosphodiester) bonds between the sugar of one nucleotide and the phosphate of the next. The energy comes from the removal of two phosphates from each incoming nucleotide.

Replication starts at many points (origins) along each eukaryotic chromosome at once, so the long molecules can be copied quickly.

Enzyme	Role
Helicase	unwinds the helix and breaks hydrogen bonds between bases
DNA polymerase	adds complementary nucleotides; forms phosphodiester bonds in the new strand

Common mistake

Saying DNA polymerase breaks hydrogen bonds or that helicase joins nucleotides. Helicase separates; polymerase builds.

Quick check

1. What does helicase do?

2. What bonds does DNA polymerase form?

Answers: 1. unwinds DNA and breaks hydrogen bonds between strands 2. covalent phosphodiester bonds between nucleotides

4 Evidence for semi-conservative replication

Heavy nitrogen showed how DNA copies.

In 1958 Matthew Meselson and Franklin Stahl tested three hypotheses: conservative (the original double helix stays intact and a completely new one is made), semi-conservative, and dispersive (old and new DNA mixed in fragments).

They grew E. coli for many generations in a medium containing the heavy isotope ¹⁵N, so all the DNA was 'heavy'. They then transferred the bacteria to a medium with normal ¹⁴N, and extracted DNA after each generation. DNA was separated by density in a centrifuge (density gradient centrifugation).

After one generation, all DNA had an intermediate density: this ruled out conservative replication (which would give one heavy and one light band). After two generations, there was one intermediate band and one light band, in equal amounts: this ruled out dispersive replication and matched the prediction of semi-conservative replication.

Generation in ¹⁴N	Semi-conservative prediction	Observed
0	all heavy	all heavy
1	all intermediate	all intermediate
2	half intermediate, half light	half intermediate, half light

Quick check

1. Why was ¹⁵N used?

2. Which result ruled out conservative replication?

Answers: 1. to make DNA heavy so new and old strands could be distinguished by density 2. a single intermediate band after one generation

5 The polymerase chain reaction (PCR)

Copying DNA millions of times in a few hours.

The polymerase chain reaction (PCR) is a laboratory technique that amplifies (makes many copies of) a specific section of DNA. It is used when only a tiny sample is available, for example from a crime scene, a fossil or a virus test.

The reaction mixture contains: the template DNA; primers (short single-stranded DNA sequences complementary to the ends of the target region); free nucleotides; and Taq polymerase, a DNA polymerase from the bacterium Thermus aquaticus, which lives in hot springs. Taq polymerase is heat-stable, so it is not denatured at the high temperatures used.

Each cycle has three steps: (1) Denaturation at about 95 °C: hydrogen bonds break and the DNA strands separate. (2) Annealing at about 55 °C: primers bind to complementary sequences at the ends of the target region. (3) Extension at about 72 °C (the optimum temperature of Taq polymerase): Taq adds nucleotides to the primers, building new complementary strands.

Each cycle doubles the number of DNA copies. After n cycles there are 2ⁿ copies of each starting molecule; 30 cycles give over a billion copies. The cycles are run automatically in a thermal cycler.

Figure 2. One cycle of PCR.

WORKED EXAMPLE 1 PCR amplification

A sample contains 50 copies of a DNA fragment. How many copies will there be after 20 cycles of PCR?

Solution

Copies = 50 × 2²⁰M1

= 50 × 1 048 576 = about 5.2 × 10⁷ copiesA1

In the real world

RT-PCR tests for SARS-CoV-2 first converted viral RNA into DNA using reverse transcriptase, then amplified it by PCR, detecting even a few viral particles in a nasal swab.

Quick check

1. Why is Taq polymerase used?

2. What happens at 55 °C?

3. How many copies after 10 cycles from one molecule?

Answers: 1. it is heat-stable 2. primers anneal to the template 3. 1 024

6 Gel electrophoresis and its applications

Separating DNA fragments by size.

Gel electrophoresis separates DNA fragments (or proteins) by size. DNA samples are loaded into wells at one end of an agarose gel, and an electric current is applied. DNA is negatively charged (because of its phosphate groups), so fragments move towards the positive electrode. Smaller fragments move faster through the pores of the gel and travel further; larger fragments travel less far. A ladder of fragments of known sizes is run alongside to estimate sizes. The DNA is stained to make bands visible.

DNA profiling (fingerprinting) uses PCR to amplify regions of the genome that vary greatly between individuals (short tandem repeats, STRs), then separates them by gel electrophoresis. Each person has a unique pattern of bands (except identical twins).

Applications: forensic investigations (matching DNA from a crime scene to a suspect); paternity testing (every band in a child's profile must come from either the mother or the father); identifying remains; and conservation and evolutionary studies.

Figure 3. A DNA profile for a paternity test: the child's bands must match the mother or the father.

WORKED EXAMPLE 2 Paternity test

Using the profile, identify the likely father and explain.

Solution

Child's band at 7.6 matches the motherR1

Child's other band (5.4) must come from the father; only father B has a band at 5.4R1

So father B is the likely fatherA1

Quick check

1. Why does DNA move towards the positive electrode?

2. Which fragments travel furthest?

Answers: 1. DNA is negatively charged (phosphate groups) 2. the smallest

7 Directionality: leading and lagging strands (HL only)

DNA polymerase can only build in one direction.

HL only

This section is Additional Higher Level content. SL students do not need it.

DNA polymerase can only add nucleotides to the 3' end of a growing strand, so new DNA is always built in the 5' to 3' direction. Because the two template strands are antiparallel (A1.2), the two new strands must be built in opposite directions.

On one template, the new strand can be built continuously towards the replication fork, following helicase: this is the leading strand. On the other template, the new strand must be built away from the fork, in short sections, each started as more of the template is exposed: this is the lagging strand. The short sections are called Okazaki fragments (about 100 to 200 nucleotides in eukaryotes).

8 The enzymes of replication and proofreading (HL only)

A team of enzymes copies DNA with very few errors.

HL only

This section is Additional Higher Level content. SL students do not need it.

Helicase unwinds and separates the strands. DNA gyrase (topoisomerase) relieves the strain caused by unwinding ahead of the fork. Single-stranded binding proteins keep the separated strands apart. DNA primase adds a short RNA primer to each template, because DNA polymerase cannot start a new strand from nothing; it can only add to an existing 3' end. DNA polymerase III adds nucleotides to the primer in the 5' to 3' direction, building the leading strand continuously and the lagging strand as Okazaki fragments. DNA polymerase I removes the RNA primers and replaces them with DNA. DNA ligase joins the Okazaki fragments by forming the final phosphodiester bonds.

Proofreading: DNA polymerase III checks each new base pair. If a mismatched nucleotide has been added, it removes it (using 3' to 5' exonuclease activity) and inserts the correct one. This reduces the error rate dramatically.

Enzyme	Role
Helicase	unwinds DNA, breaks hydrogen bonds
DNA gyrase	relieves supercoiling ahead of fork
Primase	adds RNA primers
DNA polymerase III	builds new strands 5' to 3'; proofreads
DNA polymerase I	replaces RNA primers with DNA
DNA ligase	joins Okazaki fragments

Quick check

1. Why is a primer needed?

2. What are Okazaki fragments?

3. What does DNA ligase do?

Answers: 1. DNA polymerase can only add to an existing 3' end 2. short sections of the lagging strand 3. joins Okazaki fragments

9 Why DNA must be replicated

Growth, repair and reproduction.

DNA replication produces two identical copies of the genome before cell division, so each daughter cell receives a complete set of genetic information. It is needed for growth, tissue repair and asexual and sexual reproduction.

WORKED EXAMPLE 3 Purpose (basic)

State two reasons why cells replicate their DNA.

Solution

Growth and repair (mitosis)A1

Reproduction (gamete formation)A1

10 Semi-conservative replication

One old strand, one new.

Each new DNA molecule consists of one original (parental) strand and one newly synthesized strand. Complementary base pairing ensures the new strand is an exact copy of the original partner strand.

WORKED EXAMPLE 4 Semi-conservative (standard)

Explain what is meant by semi-conservative replication.

Solution

Each daughter molecule contains one parental strandR1

and one newly synthesized strandR1

11 The Meselson–Stahl experiment

Testing the models.

Bacteria grown in 15N (heavy) were transferred to 14N (light). DNA was separated by density gradient centrifugation. After one generation, all DNA was intermediate (ruling out conservative replication); after two, half intermediate and half light (ruling out dispersive replication).

Generation	Conservative	Semi-conservative	Dispersive
0	heavy	heavy	heavy
1	heavy + light	all intermediate	all intermediate
2	1 heavy : 3 light	1 intermediate : 1 light	all one band, lighter

WORKED EXAMPLE 5 Interpreting (exam level)

Explain how the results after two generations support semi-conservative replication.

Solution

Two bands: intermediate and light in equal amountsR1

Conservative would give heavy and light; dispersive a single bandR1

Quick check

1. Predict the result after three generations.

Answers: 1. 1 intermediate : 3 light

12 Helicase and unwinding

Separating the strands.

Helicase unwinds the double helix and breaks the hydrogen bonds between complementary bases, separating the two strands and forming a replication fork. Single-stranded binding proteins keep the strands apart.

WORKED EXAMPLE 6 Helicase (standard)

State the role of helicase.

Solution

Unwinds DNA and breaks hydrogen bonds between basesA1

13 DNA polymerase and complementary pairing

Building new strands.

Free nucleotides (as deoxynucleoside triphosphates) pair with exposed bases: A with T, C with G. DNA polymerase links them by covalent phosphodiester bonds, adding nucleotides to the 3′ end of the growing strand, so synthesis is always 5′→3′.

WORKED EXAMPLE 7 Direction (standard)

Explain why DNA polymerase synthesizes DNA only 5′→3′.

Solution

It can only add nucleotides to the free 3′ OH groupR1

14 Leading and lagging strands

Continuous and discontinuous synthesis.

Because the strands are antiparallel and synthesis is 5′→3′, one strand (leading) is synthesized continuously towards the fork; the other (lagging) is synthesized discontinuously in short Okazaki fragments away from the fork, later joined by DNA ligase.

WORKED EXAMPLE 8 Lagging strand (exam level)

Explain why the lagging strand is synthesized in fragments.

Solution

Strands antiparallel; polymerase works only 5′→3′R1

Lagging strand must be made away from the fork in short pieces (Okazaki fragments)R1

Joined by DNA ligaseR1

15 Primers and primase

Getting started.

DNA polymerase cannot start a new strand; it needs a short RNA primer, made by primase. On the leading strand one primer is needed; on the lagging strand each Okazaki fragment needs a primer. Primers are later removed and replaced with DNA.

Quick check

1. Why does the lagging strand need many primers?

Answers: 1. each Okazaki fragment must be started separately

16 Proofreading and accuracy

Error rates.

DNA polymerase III proofreads: if an incorrect base is added, it removes it (3′→5′ exonuclease activity) and adds the correct one. This reduces errors to about one in 109–1010 bases.

WORKED EXAMPLE 9 Errors (standard)

Estimate the number of errors when replicating the human genome (3.2 × 109 bp) at an error rate of 1 in 109.

Solution

3.2 × 109/109M1

= about 3 errors per replicationA1

17 The polymerase chain reaction (PCR)

Copying DNA in vitro.

PCR amplifies DNA through repeated cycles: denaturation (about 95 °C, strands separate), annealing (about 55 °C, primers bind), and extension (about 72 °C, Taq polymerase adds nucleotides). Each cycle doubles the DNA.

WORKED EXAMPLE 10 Copies (standard)

How many copies of a DNA sequence are produced from one molecule after 25 cycles?

Solution

225M1

= 33 554 432 (about 3.4 × 107)A1

In the real world

Rapid PCR tests detected SARS-CoV-2 RNA (after reverse transcription) during the COVID-19 pandemic.

18 Taq polymerase

Heat-stable enzyme.

Taq polymerase comes from Thermus aquaticus, a bacterium from hot springs. It does not denature at 95 °C, so it does not need to be replaced each cycle, making automated PCR possible.

Quick check

1. Why was Taq polymerase important for PCR?

Answers: 1. heat stable: survives denaturation step

19 Gel electrophoresis

Separating DNA fragments.

DNA fragments are loaded into wells in an agarose gel. An electric field moves the negatively charged DNA towards the positive electrode; smaller fragments move faster and further. Fragment sizes are estimated by comparison with a DNA ladder.

WORKED EXAMPLE 11 Separation (standard)

Explain how DNA fragments are separated.

Solution

DNA negatively charged (phosphate groups); moves to positive electrodeR1

Smaller fragments move further through the gelR1

20 DNA profiling

Applications.

DNA profiling compares short tandem repeats (STRs) at several loci. Uses: forensic identification, paternity testing, identifying remains, and wildlife conservation. Matching all bands indicates the same individual; half of bands from each parent in paternity tests.

WORKED EXAMPLE 12 Paternity (exam level)

How are DNA profiles used in paternity testing?

Solution

Child's bands come half from mother, half from fatherR1

Bands not from the mother must match the alleged fatherR1

21 Mixed review: worked problems

Bringing the ideas together.

WORKED EXAMPLE 13 Mixed problem (exam level)

Compare DNA replication in cells with PCR.

Solution

Both: complementary base pairing, DNA polymerase, primersR1

Cells: helicase separates strands; PCR: heatR1

PCR: Taq polymerase, short target sequence, repeated cyclesR1

22 Additional exam-style practice

Paper 1 and Paper 2.

WORKED EXAMPLE 14 Paper 1 question (standard)

Which enzyme joins Okazaki fragments? A. helicase B. primase C. DNA ligase D. DNA polymerase III

Solution

CA1

WORKED EXAMPLE 15 Paper 2 question (exam level)

Outline the stages of a PCR cycle.

Solution

Denaturation at about 95 °CR1

Annealing of primers at about 55 °CR1

Extension by Taq polymerase at about 72 °CR1

23 Replication in prokaryotes and eukaryotes

Origins and speed.

Prokaryotes have one origin of replication on their circular chromosome. Eukaryotes have many origins on each linear chromosome, allowing their large genomes to be replicated in a reasonable time.

Quick check

1. Why do eukaryotic chromosomes have many origins of replication?

Answers: 1. large genomes would take too long to copy from one origin

24 Telomeres

Protecting chromosome ends.

Telomeres are repetitive sequences at chromosome ends. The lagging strand cannot be fully replicated at the end, so telomeres shorten with each division, protecting genes. Telomerase extends telomeres in stem cells and many cancer cells.

Quick check

1. Why do telomeres shorten with each cell division?

Answers: 1. the lagging strand cannot be completed at the chromosome end

25 Practical: modelling replication

Paper and bead models.

Building physical models of DNA replication (paper strips or beads) helps visualize antiparallel strands, the replication fork, leading and lagging strands, and Okazaki fragments.

WORKED EXAMPLE 16 Model (standard)

What does the model show about the lagging strand?

Solution

It must be built in short sections away from the forkR1

Try this

Use two coloured paper strips labelled 5′ and 3′ to model the replication fork, adding new nucleotides only in the 5′→3′ direction.

26 Common misconceptions and links

Students often think…	Actually…
replication is conservative	it is semi-conservative
helicase builds DNA	helicase separates strands; polymerase builds
large fragments move furthest in gels	small fragments move furthest
both strands are built continuously	the lagging strand is built as Okazaki fragments

How D1.1 connects to the rest of the course

Topic	Connection
A1.2 Nucleic acids	structure and base pairing
D2.1 Cell division	S phase
D1.3 Mutations	replication errors
D1.2 Protein synthesis	transcription
A3.1 Diversity	DNA sequencing

27 Exam-style question with markscheme

Try this under exam conditions (about 18 minutes) before reading the markscheme.

Figure 4. Meselson-Stahl results.

(a) Explain the result after one generation in ¹⁴N. [2]

(b) Predict the result after three generations. [1]

(d) Explain why small DNA fragments travel further in gel electrophoresis. [2]

(e) Explain why the lagging strand is built in fragments. [3]

Markscheme

Part	Answer	Marks
(a)	each molecule has one ¹⁵N and one ¹⁴N strand	R1
	intermediate density; rules out conservative	R1
(b)	1/4 intermediate, 3/4 light	A1
(c)	denaturation at 95 °C	A1
	annealing of primers at 55 °C	A1
	extension by Taq at 72 °C	A1
(d)	move more easily through gel pores	R1
	DNA negatively charged, moves to positive electrode	R1
(e)	polymerase only builds 5' to 3'	R1
	strands antiparallel	R1
	lagging strand built away from fork in short Okazaki fragments joined by ligase	R1

Examiner's comments. In (a), the best answers explained why the result ruled out conservative replication.

28 Check your understanding

Section A: multiple choice

1. Replication is semi-conservative because

A. each new molecule contains one original strand B. both strands are new C. the original molecule is kept intact D. DNA is mixed randomly

2. Helicase

A. adds nucleotides B. joins fragments C. breaks hydrogen bonds between bases D. makes primers

3. Taq polymerase is used in PCR because it is

A. cheap B. heat-stable C. fast D. from humans

4. In PCR, annealing is at about

A. 95 °C B. 72 °C C. 37 °C D. 55 °C

5. In gel electrophoresis DNA moves to the

A. negative electrode B. positive electrode C. top D. wells

6. After 3 PCR cycles one DNA molecule becomes

A. 8 molecules B. 3 C. 6 D. 9

7. Okazaki fragments are joined by

A. helicase B. primase C. DNA polymerase III D. DNA ligase

8. RNA primers are removed by

A. ligase B. helicase C. DNA polymerase I D. primase

Section B: structured

9. Explain semi-conservative replication. [4]

10. Describe how the Meselson-Stahl experiment supported it. [4]

11. Explain how DNA profiling is used in paternity testing. [3]

12. Explain the roles of primase, DNA polymerase I and ligase. [3]

Answers

1. A 2. C 3. B 4. D 5. B 6. A 7. D 8. C

9. Strands separate (helicase); each a template; complementary nucleotides added by polymerase; each new molecule half old.

10. ¹⁵N then ¹⁴N; intermediate after one generation; intermediate + light after two.

11. STRs amplified; child bands from mother or father; all non-maternal bands must match the father.

12. Primase: RNA primer; Pol I: replaces primers with DNA; ligase: joins fragments.

29 Summary and topic map

Idea	Remember
Purpose	reproduction, growth, repair
Semi-conservative	one old, one new strand; complementary pairing
Enzymes	helicase separates; DNA polymerase builds
Evidence	Meselson-Stahl with ¹⁵N
PCR	95, 55, 72 °C; Taq; doubling
Gel electrophoresis	small fragments travel furthest; profiling
HL	5' to 3'; leading and lagging strands; primase, Pol III, Pol I, ligase; proofreading

Before the exam, make sure you…

•name enzymes precisely
•give PCR temperatures and steps
•interpret gels band by band
•predict Meselson-Stahl results

DNA Replication on one page

Cover this page with a sheet of paper and try to rebuild it from memory. Whatever you can't recall is what to revise next.

Figure 5. Topic map for DNA Replication.

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