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PCR

Laura Armstrong & Joe Wolfensohn

Teachers

Laura Armstrong Joe Wolfensohn

Contents

What is PCR?

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PCR vs In Vivo DNA Replication

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Recall Questions

This topic requires prior knowledge of DNA replication from Year 12. You can test your knowledge on this below.

What enzyme joins adjacent DNA nucleotides during DNA replication?

DNA polymerase.

Which enzyme breaks hydrogen bonds between DNA strands?

DNA helicase.

What is meant by semi-conservative replication?

Each new DNA molecule contains one original strand and one newly synthesised strand.

Topic Explainer Video

Check out this @JoeDoesBiology video that explains PCR or read the full notes below. Once you've gone through the whole note, try out the practice questions!

What is PCR?

The Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify DNA - producing millions of copies of a specific DNA fragment in just a few hours.

PCR is used in:

  • Medical diagnostics (e.g. COVID-19 tests, cancer detection)

  • Forensics (DNA fingerprinting)

  • Genetic research and cloning

  • Detecting mutations or pathogens

For PCR, you require:

  • The DNA fragment to be amplified

  • Nucleotides of all 4 types, A, T, C and G

  • Primers

  • DNA polymerase

 

PCR Steps

PCR mimics DNA replication, but occurs in vitro (outside of a living cell). It involves three main steps:

  • Denaturation 

    • DNA is heated to break hydrogen bonds between strands → DNA becomes single-stranded.

  • Annealing 

    • Short DNA primers bind (anneal) to complementary sequences at the ends of the target DNA.

  • Synthesis

    • Taq DNA polymerase (heat-stable enzyme from Thermus aquaticus bacteria) joins free nucleotides to the primers → forming new DNA strands by making phosphodiester bonds.

    • 72°C is used as this is the optimum temperature for this type of DNA Polymerase.

    • As this enzyme is thermostable it will not be denatured by the high temperatures used in PCR to separate the DNA strands.

     

This cycle is repeated multiple times, doubling the DNA each time.

Role of Primers in PCR

Primers are short, single-stranded DNA sequences (usually about 18–25 bases long) that are complementary to the start of the target DNA region to be amplified.

PCR uses two primers:

  • One has a complementary base sequence to the start of one DNA strand

  • The other has a complementary base sequence to the start of the other DNA strand

They have two key roles:

1. Provide a Starting Point for DNA Polymerase

    • Taq DNA polymerase (used in PCR) cannot start building a new DNA strand from scratch.

    • It can only add nucleotides to an existing strand - primers provide this essential starting point.

    • Once a primer is bound (annealed), Taq polymerase can extend the strand by joining DNA nucleotides together.

2. Prevent the original strands from rejoining

When DNA is heated during PCR (around  ), the hydrogen bonds between the complementary bases are broken, so the two strands separate.

  • If left alone, as the mixture cools, the strands would naturally reanneal (reform hydrogen bonds) because they are perfectly complementary.

  • As the mixture cools to  , primers are present in high concentration.

  • Because primers are shorter than the original DNA strands, they anneal (bind) faster.

  • This physically blocks the original complementary strands from rejoining.

 

Calculating DNA Fragments in PCR

After n cycles, the number of DNA fragments = 2n.

Example:

After 5 cycles

DNA fragments = 25

= 32 fragments of DNA

 

Why Might PCR Stop or be Inefficient?

  1. Primers or nucleotides may run out.

  2. Taq polymerase may eventually denature over time.

  3. Temperature damages some of the DNA fragments.

  4. Once separated, the original DNA strands may re-join (rather than bonding to primers).

 

PCR vs In Vivo DNA Replication

Feature

PCR (In Vitro)

In Vivo DNA Replication

Location

In a machine (thermocycler)

Inside the cell (nucleus)

Enzymes Used

Taq DNA polymerase (thermostable)

DNA polymerase (body temp-sensitive)

DNA Primer Requirement

Yes – artificial short primers needed

DNA primers not required

DNA Unwinding

By heat (no helicase)

By DNA helicase

Number of Copies

Exponential   with each cycle

One copy per cell cycle

Application

Used in biotechnology and diagnostics

Used in cell division

Key Terms

  • PCR: A method used to amplify DNA in vitro using repeated cycles of heating and cooling.

  • Primer: Short, single stranded DNA that binds to the template strand to allow polymerase binding.

  • Taq DNA Polymerase: A heat-stable enzyme that synthesises new DNA strands.

  • Annealing: Binding of primers to single-stranded DNA via complementary base pairing.

No answer provided.

Exam Tip

Learn the process in steps and explain the importance of each one:

  • Denaturation: 

  • Annealing: 

  • Synthesis: 

Use the phrase “doubles the amount of DNA per cycle” and “exponential increase” when describing amplification.

No answer provided.

Describe how the polymerase chain reaction is used to amplify a DNA fragment. (5 marks)

  • DNA fragment, nucleotides, primers and DNA polymerase are required.

  • DNA is heated to    to break hydrogen bonds and separate strands.

  • The mixture is cooled to  , allowing primers to anneal to the DNA.

  • Temperature is raised to  , where DNA polymerase synthesises the DNA by joining nucleotides together.

  • This cycle of heating and cooling is repeated, doubling the DNA each time.

Practice Question 1

Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!

Practice Question 2

Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!

Electrophoresis