DNA Replication

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Semi-Conservative Replication

DNA replication is semi-conservative: each new DNA molecule contains one original (parent) strand and one newly synthesised strand.
The original strand is called the template strand; the new strand is built from free nucleotides by complementary base pairing.
Base pairing rules: adenine pairs with thymine, and cytosine pairs with guanine.
Hydrogen bonds form only between correctly paired bases on the template and new strands.
Replication occurs in the nucleus and is important for growth, replacement of old/damaged cells, and reproduction.
Crick and Watson proposed the semi-conservative model; Meselson and Stahl later provided experimental evidence.

The Role of Helicase and DNA Polymerase

Helicase unwinds the DNA double helix and breaks the hydrogen bonds between base pairs, exposing bases on each strand.
Each single strand acts as a template for the formation of a new strand.
DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template.
DNA polymerase catalyses condensation reactions between the deoxyribose sugar and phosphate groups of adjacent nucleotides, forming the sugar-phosphate backbone.
DNA polymerase always builds the new strand in the 5' to 3' direction, attaching to the 3' end of the template strand and reading it in a 3' to 5' direction.

Directionality and Strand Synthesis

DNA polymerase only works in a 5' to 3' direction, adding nucleotides to the 3' end of a growing strand.
The 5' phosphate group of an incoming nucleotide bonds to the free 3'-OH group on the growing strand.
Double-stranded DNA consists of two antiparallel strands: one runs 5' to 3', the other 3' to 5'.
The leading strand is synthesised continuously, following the replication fork as it opens.
The lagging strand is synthesised discontinuously in short fragments called Okazaki fragments, away from the fork.
Okazaki fragments are later joined together by DNA ligase to form a continuous strand.

Enzymes in DNA Replication

Helicase unwinds the double helix and breaks hydrogen bonds between strands at the replication fork.
Single-stranded binding proteins keep the separated strands apart while the template is copied.
DNA primase generates a short RNA primer on the template strands, providing an initiation point for DNA polymerase III.
DNA polymerase III starts replication next to the RNA primer, linking nucleotides in a 5' to 3' direction.
DNA polymerase I removes the RNA primers and replaces them with DNA.
DNA ligase joins Okazaki fragments by catalysing the formation of sugar-phosphate bonds.
An RNA primer is needed once on the leading strand but several times on the lagging strand to initiate each fragment.

Proofreading and Accuracy

Each human cell replication requires synthesis of about 3 billion new base pairs.
Mistakes during replication are called mutations and can be harmful, leading to diseases such as cancer.
In prokaryotes, DNA polymerase III acts as a proof-reader of the new daughter strand.
It recognises incorrect nucleotides, reverses direction to remove the incorrect nucleotide from the 3' end, then inserts the correct one and continues replication.
Semi-conservative replication ensures genetic continuity with high accuracy between generations of cells.

Gel Electrophoresis

Gel electrophoresis separates DNA, RNA, and proteins according to their size (mass) and net charge.
DNA is negatively charged due to its phosphate groups, so it moves towards the anode (positive pole).
Smaller molecules move faster and further through the gel pores; larger molecules move more slowly.
Agarose gel is used for DNA; polyacrylamide gel is used for proteins.
Method: create an agarose gel plate with wells, submerge it in electrolyte solution, load DNA fragments into wells, and apply an electric current.
The negative electrode must be connected to the end with the wells so DNA fragments move towards the anode.
Fragments are visualised using radioactive labels (X-ray film) or fluorescent stains (UV light).

Polymerase Chain Reaction (PCR)

PCR is an in vitro method of DNA amplification used to produce large quantities of specific DNA or RNA fragments from very small samples.
Requirements: target DNA/RNA, DNA polymerase (commonly Taq polymerase), free nucleotides, buffer solution, and primer sequences.
Taq polymerase comes from the thermophilic bacterium×Thermus aquaticus×and does not denature at high temperatures.
PCR occurs in a thermal cycler and involves three stages per cycle.
Denaturation: heated to 95°C to break hydrogen bonds between DNA strands.
Annealing: cooled to 50–60°C so primers bind to the single strands.
Elongation/Extension: heated to 72°C for Taq polymerase to build complementary strands.
Each cycle doubles the DNA; 30 cycles can generate over 1 billion copies by exponential amplification.

DNA Profiling and Its Applications

DNA profiling (genetic fingerprinting) identifies suspects, victims, and ancestry relationships.
The regions analysed are VNTRs (Variable Number Tandem Repeats): short, non-coding repeating DNA sequences 20–50 bases long.
Every person (except identical twins) has a unique combination of VNTRs.
Steps: DNA extraction, PCR amplification, cutting with restriction endonucleases, gel electrophoresis, adding probes, and visualisation.
In paternity testing, a parent shares about 50% of the offspring's bands; any band in the child must be present in the mother or father.
In forensics, samples from a crime scene are compared to suspects, victims, and control samples; care must be taken to avoid contamination.
Reliability is increased by using more VNTR markers, reducing the chance of a false match.

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Practice questions

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1.Which of the following correctly describes the role of DNA polymerase in DNA replication?
Easy
- AForms hydrogen bonds between the bases of the new nucleotides and the template strand.
- BUnwinds the DNA double helix at the replication fork.
- CCovalently bonds new DNA nucleotides together.
- DJoins up the Okazaki fragments on the lagging strand.
2.To which part of the DNA molecule will new nucleotides be covalently bonded during DNA replication?
Easy
- ANitrogenous base
- B5’ phosphate group of a pentose sugar
- C1’ carbon of a pentose sugar
- D3’ -OH group of a pentose sugar
3.In gel electrophoresis, DNA moves towards the _________.
Easy
- ACathode
- BNegatively charged electrode
- CAnode
- DTiny pores in the gel
4.Which of the following processes takes place in a thermal cycler?
Easy
- AGenetic modification
- BPolymerase chain reaction
- CGel electrophoresis
- DGenetic fingerprinting
5.Which of the following statements about semi-conservative replication of DNA are correct? (select all that apply)
Medium
- ADNA polymerase always builds a new strand of DNA in the 3′ to 5′ direction.
- BThe action of DNA polymerase is followed by that of helicase.
- COne strand of original DNA remains in each daughter molecule; the other strand has been made from free nucleotides.
- DCrick and Watson proposed the hypothesis of semi-conservative replication, whereas experimental evidence was later provided by Meselson and Stahl.
6.During the process of semi-conservative replication of DNA, which activity relating to bonding does not take place?
Medium
- ABreaking of hydrogen bonds.
- BDNA polymerase forms an enzyme-substrate complex with free nucleotides.
- CFormation of new covalent bonds between nucleotides.
- DFormation of new glycosidic bonds between deoxyribose molecules.
7.Which of the following are necessary for successfully conducting PCR in a forensic examination at a crime scene? (select all that apply)
Medium
- AAn active sample of human DNA or RNA polymerase
- BBuffer solution
- CA sample of target DNA or RNA
- DFree nucleotides
- EPrimer sequences
8.Okazaki fragments are short sections of DNA formed during DNA replication. Which of the following statements correctly describes why they form?
Easy
- ATo help the DNA helix unwind
- BTo enable replication of the leading strand
- CTo act as a template for DNA polymerase
- DTo enable replication of the lagging strand

More questions

9.Which of the following statements explains the separation of molecules in electrophoresis?
Easy
- ADNA molecules from eukaryotes are broken up into smaller fragments.
- BNegatively charged proteins travel towards the anode.
- CPositively charged DNA molecules travel towards the cathode.
- DThe larger the molecule, the further it travels through the gel.
10.What is the correct order of events for the stages of PCR?
Medium
- AAnnealing, Elongation, Denaturation
- BDenaturation, Annealing, Elongation
- CDenaturation, Elongation, Annealing
- DElongation, Annealing, Denaturation
11.What key feature of the genetic code makes it possible to transfer genes between species?
Easy
- AIt has 2 strands.
- BIt is universal.
- CThe code is degenerate.
- DIt can be amplified using PCR.
12.Which of the following shows the correct order of events in DNA replication?
Medium
- AComplementary strands are separated → hydrogen bonds form between complementary bases → hydrogen bonds break between complementary bases → free DNA nucleotides pair with complementary nucleotides → covalent bonds form between the pentose sugar and phosphate group of adjacent nucleotides
- BHydrogen bonds form between complementary bases → complementary strands are separated → free DNA nucleotides pair with complementary nucleotides → hydrogen bonds break between complementary bases → covalent bonds form between the pentose sugar and phosphate group of adjacent nucleotides
- CFree DNA nucleotides pair with complementary nucleotides → complementary strands are separated → hydrogen bonds form between complementary bases → hydrogen bonds break between complementary bases → covalent bonds form between the pentose sugar and phosphate group of adjacent nucleotides
- DHydrogen bonds break between complementary bases → complementary strands are separated → free DNA nucleotides pair with complementary nucleotides → hydrogen bonds form between complementary bases → covalent bonds form between the pentose sugar and phosphate group of adjacent nucleotides
13.Match each enzyme with its role in DNA replication.
Medium
- Helicase
- DNA polymerase III
- DNA ligase
- DNA primase
- Joins Okazaki fragments by catalysing formation of sugar-phosphate bonds
- Unwinds the DNA double helix and breaks hydrogen bonds
- Generates a short RNA primer on the template strand
- Starts replication next to the RNA primer, linking nucleotides in a 5’ to 3’ direction
14.Place the stages of one PCR cycle in the correct order.
Medium
- Annealing
- Denaturation
- Elongation
15.DNA replication is semi-conservative because one strand of the parent DNA is kept in each daughter molecule.
Easy
True or false?
16.DNA polymerase builds new strands in the 3' to 5' direction.
Easy
True or false?
17.Which of the following statements about the requirements of PCR are correct? (select all that apply)
Medium
- ABuffer solution is needed to provide the optimum pH for the reaction
- BTaq polymerase is used because it does not denature at high temperatures
- CFree nucleotides are required to build the new DNA strands
- DDNA ligase is essential for joining the newly synthesised strands
- EPrimers are required to provide a starting point for DNA polymerase
18.Match each enzyme with its function in DNA replication.
Medium
- Helicase
- DNA polymerase III
- DNA ligase
- DNA primase
- Joins Okazaki fragments by forming sugar-phosphate bonds
- Unwinds the double helix and breaks hydrogen bonds between strands
- Synthesises new DNA strands by adding nucleotides in a 5' to 3' direction
- Generates a short RNA primer to initiate DNA synthesis
19.Place the following events of DNA replication in the correct order.
Medium
- Helicase unwinds the double helix and breaks hydrogen bonds
- Single-stranded binding proteins keep the separated strands apart
- DNA primase synthesises a short RNA primer
- DNA polymerase III adds DNA nucleotides in a 5' to 3' direction
- DNA ligase joins Okazaki fragments
20.Which of the following are true about the leading and lagging strands during DNA replication? (select all that apply)
Hard
- AThe leading strand is synthesised continuously in the same direction as the replication fork
- BThe lagging strand is synthesised discontinuously in short Okazaki fragments
- CDNA polymerase III adds nucleotides to the 3' end of the growing strand
- DThe lagging strand is synthesised in a 3' to 5' direction
- EOkazaki fragments are joined together by DNA ligase
21.DNA polymerase always builds a new DNA strand in the 5' to 3' direction.
Easy
True or false?
22.During PCR, the annealing step occurs at 72°C.
Easy
True or false?
23.Which of the following correctly describes the role of DNA ligase in DNA replication?
Medium
- AIt unwinds the double helix at the replication fork.
- BIt joins Okazaki fragments by catalysing the formation of sugar-phosphate bonds.
- CIt synthesises RNA primers to initiate DNA synthesis.
- DIt proofreads the newly synthesised DNA strand and removes incorrect nucleotides.
24.In gel electrophoresis, why does DNA move towards the anode?
Medium
- ADNA is positively charged due to its phosphate groups.
- BDNA is negatively charged due to its phosphate groups.
- CDNA is attracted to the negative electrode.
- DDNA moves randomly through the gel pores.
25.Which of the following is the correct order of the three stages of one PCR cycle?
Medium
- AAnnealing → Denaturation → Elongation
- BDenaturation → Annealing → Elongation
- CElongation → Annealing → Denaturation
- DDenaturation → Elongation → Annealing
26.The diagram shows the outcomes from four different models of DNA replication after one nuclear division. The parent DNA is shown in black and the newly synthesised DNA is shown in grey. Which diagram represents semi-conservative DNA replication?
Medium
- ADiagram 1: one molecule has two black strands, the other has two grey strands
- BDiagram 2: both molecules each contain one black strand and one grey strand
- CDiagram 3: both molecules contain a mixture of black and grey segments along each strand
- DDiagram 4: one molecule has one black and one grey strand, the other has two grey strands
27.The DNA profile shows the banding patterns of a child (X), a mother, and three possible fathers. Which two individuals could be the parents of child X?
Medium
- AMother and male 1
- BMother and male 2
- CMother and male 3
- DMale 1 and male 3
28.The shaded boxes in a diagram indicate VNTR sequences from three loci amplified by PCR from DNA found at a crime scene. The DNA profiles of four suspects are also shown. Which suspect was most likely at the crime scene?
Medium
- ASuspect 1
- BSuspect 2
- CSuspect 3
- DSuspect 4
29.Which statement cannot be concluded from a DNA profile showing two parents and several children?
Medium
- AParent 1 and parent 2 are profiles of unrelated individuals.
- BChildren 3 and 5 are non-identical twins.
- CChild 4 is related to parent 1 but not parent 2.
- DChild 6 is unrelated to any of the other individuals.
30.A DNA profile was created for two genes in three individuals (A, B, and C). Which of the following statements, relating to the images provided, are correct? I. The relationship between DNA fragment length and distance moved through the gel is linear. II. Fragment length is determined by the number of repeated DNA sequences in a sample. III. There is a percentage increase of 50% between the distance moved by fragment Y and that moved by fragment X. IV. Individual A is homozygous for both of the genes tested.
Medium
- AI, II, and III only
- BI, II, III, and IV
- CII and IV only
- DIII and IV only
31.A DNA sample is amplified by PCR for 6 cycles. If you started with one double-stranded DNA molecule, how many double-stranded DNA molecules will be present after 6 cycles?
Hard
- A12
- B36
- C64
- D128
32.A forensic scientist uses gel electrophoresis to separate DNA fragments. Fragment A is 200 base pairs long and fragment B is 800 base pairs long. Which statement correctly describes their movement through the gel?
Hard
- AFragment A moves faster and further towards the anode than fragment B.
- BFragment B moves faster and further towards the anode than fragment A.
- CFragment A moves faster and further towards the cathode than fragment B.
- DBoth fragments move at the same speed because they have the same charge.
33.A scientist performs PCR but forgets to add the primer sequences. What is the most likely outcome?
Medium
- AThe DNA will be amplified normally because primers are not essential.
- BThe DNA strands will not separate during denaturation.
- CDNA polymerase will not have a starting point to begin synthesis.
- DThe Taq polymerase will denature at 95°C.
34.In a DNA profiling experiment, the reliability of a match between a crime scene sample and a suspect is increased by:
Hard
- AUsing a single VNTR marker
- BIncreasing the number of VNTR markers compared
- CRunning the gel at a higher voltage
- DUsing a larger sample of DNA
35.Which of the following statements about the leading and lagging strands during DNA replication are correct? (select all that apply)
Hard
- AThe leading strand is synthesised continuously in the 5' to 3' direction.
- BThe lagging strand is synthesised discontinuously as Okazaki fragments.
- CDNA ligase joins the Okazaki fragments on the lagging strand.
- DThe leading strand requires multiple RNA primers.
- EThe lagging strand is synthesised in the 3' to 5' direction.
36.A student sets up a PCR reaction. Which components are essential for the reaction to work? (select all that apply)
Hard
- ATarget DNA
- BTaq polymerase
- CFree nucleotides
- DPrimers
- ERestriction endonucleases
37.In gel electrophoresis, DNA fragments move towards the anode because DNA is negatively charged.
Easy
True or false?
38.In gel electrophoresis, what determines the rate at which a DNA fragment moves through the gel?
Medium
- AThe size of the fragment and the type of gel used
- BThe temperature of the electrolyte solution only
- CThe length of time the electric current is applied
- DThe concentration of DNA in the sample
39.A scientist carries out gel electrophoresis but connects the positive electrode to the end of the gel with the wells. What is the most likely result?
Medium
- ADNA fragments will move towards the negative electrode and be lost from the gel
- BDNA fragments will not move because they are not attracted to the positive electrode
- CDNA fragments will move in the opposite direction, away from the positive electrode
- DDNA fragments will separate normally but the bands will be inverted
40.Which of the following are necessary for the polymerase chain reaction to work? (select all that apply)
Medium
- ADNA polymerase
- BFree nucleotides
- CBuffer solution
- DRestriction enzymes
- EPrimer sequences
41.If one molecule of DNA goes through 5 PCR cycles in the thermocycler, how many copies of that molecule of DNA will be produced?
Medium
42.Complete the sentence about PCR.
Medium
The enzyme most commonly used in PCR is ____ polymerase, which comes from the thermophilic bacterium ____.
43.A sample of DNA undergoes 4 cycles of PCR. How many copies of the original DNA molecule will be produced?
Medium
44.Complete the sentence about the stages of PCR.
Medium
In PCR, the stage at which the double-stranded DNA is heated to 95°C to break hydrogen bonds is called ____, and the stage at which primers bind to the single strands is called ____.
45.A single DNA molecule is amplified by PCR. How many copies of the DNA molecule will be produced after 5 cycles?
Medium
46.In a PCR experiment, the temperature is held at 95°C for 30 seconds, then lowered to 55°C for 30 seconds, then raised to 72°C for 60 seconds. This is one cycle. If this cycle is repeated 10 times, how many copies of the original DNA molecule will be present?
Hard
47.A single double-stranded DNA molecule is amplified by PCR. How many double-stranded DNA molecules will be present after 6 cycles?
Medium
48.A PCR reaction starts with 3 double-stranded DNA molecules. How many double-stranded DNA molecules will be present after 4 cycles?
Medium
49.Complete the sentence about DNA replication.
Medium
During DNA replication, the enzyme ____ unwinds the double helix and breaks the hydrogen bonds between the strands, while the enzyme ____ joins Okazaki fragments together.
50.Name the enzyme that joins Okazaki fragments together during DNA replication.
Medium
51.Which direction does DNA polymerase build a new DNA strand?
Medium
52.If a single double-stranded DNA molecule is amplified by PCR for 4 cycles, how many double-stranded DNA molecules will be present?
Medium
53.Explain why DNA replication is described as semi-conservative.
Hard
54.Describe the role of DNA ligase in DNA replication.
Hard
55.Explain how the structure of DNA allows semi-conservative replication to occur with high accuracy.
Hard
56.Explain why the polymerase chain reaction requires a DNA polymerase from a thermophilic bacterium such as Thermus aquaticus.
Hard
57.If one molecule of DNA goes through 5 PCR cycles in the thermocycler, how many copies of that molecule of DNA will be produced?
Medium
- A29 copies
- B32 copies
- C38 copies
- D35 copies
58.A sample of DNA undergoes 4 cycles of PCR. How many copies of the original DNA molecule will be produced?
Medium
- A18 copies
- B16 copies
- C15 copies
- D17 copies
59.A single DNA molecule is amplified by PCR. How many copies of the DNA molecule will be produced after 5 cycles?
Medium
- A29 copies
- B38 copies
- C32 copies
- D35 copies
60.In a PCR experiment, the temperature is held at 95°C for 30 seconds, then lowered to 55°C for 30 seconds, then raised to 72°C for 60 seconds. This is one cycle. If this cycle is repeated 10 times, how many copies of the original DNA molecule will be present?
Hard
- A922 copies
- B1228 copies
- C1024 copies
- D1126 copies
61.A single double-stranded DNA molecule is amplified by PCR. How many double-stranded DNA molecules will be present after 6 cycles?
Medium
- A76 molecules
- B64 molecules
- C58 molecules
- D70 molecules
62.A PCR reaction starts with 3 double-stranded DNA molecules. How many double-stranded DNA molecules will be present after 4 cycles?
Medium
- A43 molecules
- B58 molecules
- C48 molecules
- D53 molecules
63.If a single double-stranded DNA molecule is amplified by PCR for 4 cycles, how many double-stranded DNA molecules will be present?
Medium
- A18 molecules
- B15 molecules
- C17 molecules
- D16 molecules

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