19.1.819 · Genetic Technology

Polymerase Chain Reaction — Cambridge AS & A Level Biology 9700 Revision Notes & Topical Questions

5 min read 9 parts Lesson 9 of 20 1 quiz

📚 Cambridge AS & A Level Biology 9700 Hub / Topical Revision

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19.1.8 Polymerase Chain Reaction — CAMBRIDGE AS & A LEVEL BIOLOGY 9700 — REVISION NOTES notes: Describe the requirements and stages of PCR and why Taq polymerase is used.PCR — an in vitro method of DNA amplification.It produces large quantities of specific DNA or RNA fragments fr…

Primers (forward and reverse)
Denaturation
Elongation
Thermal cycler
Recombinant
Electrophoresis

Part 1 of 7Purpose of PCR

PCR

PCR an in vitro method of DNA amplification.

It produces large quantities of specific DNA or RNA fragments from very small quantities, even one molecule.

Billions of identical copies can be made within a few hours.

It is used in DNA profiling (identifying criminals, paternity) and in genetic engineering.

Each cycle doubles the DNA, so 20 cycles produce about a million molecules (copies = N₀ × 2ⁿ).

The reaction is run in a thermal cycler, which controls temperature and time at each stage.

Part 2 of 7Requirements

Requirements

Target DNA or RNA being amplified.

Primers (forward and reverse)

Primers (forward and reverse) short single-stranded DNA sequences complementary to the 3' end of the sequence being copied.
They define the region to be amplified by signalling where polymerase starts.

DNA polymerase, usually Taq polymerase.

Free nucleotides to build the new strands.

Buffer solution to give the optimum pH.

Part 3 of 7Stages of each cycle

Stage	Temperature	What happens
Denaturation	95 °C	Hydrogen bonds between the two DNA strands break
Annealing	50-60 °C	Forward and reverse primers anneal to the ends of the single strands
Elongation (extension)	72 °C for at least a minute	Taq polymerase builds complementary strands to give new identical double-stranded DNA

Part 4 of 7Why Taq polymerase is used

Taq polymerase comes from the thermophilic bacterium Thermus aquaticus.

It does not denature at the high temperature of the first stage.

Its optimum temperature (72 °C) is high enough to prevent annealing of strands not yet copied.

No new enzyme needs adding each cycle, so the process can be automated.

Part 5 of 7Choice of conditions

Choice of conditions

Annealing must be cool enough for primers to bind but warm enough to prevent non-specific binding.

Primers must not bind to each other and should have similar annealing temperatures.

Each product is used as a template in the next cycle, hence "chain reaction".

Part 6 of 7Uses and limitations

Uses

Uses forensic identification, detecting pathogen DNA, screening for mutations and amplifying genes for cloning and sequencing.

Limitations:

Contamination with foreign DNA is also amplified.
Sequence information is needed to design primers.
Taq polymerase lacks proofreading so occasional errors occur.
Only relatively short sequences are copied efficiently.

Part 7 of 7More from the specification

Restriction endonucleases in recombinant technology

Restriction endonucleases in recombinant technology scientists use restriction endonucleases to excise desired genes from donor organisms for insertion into bacterial plasmids.

Reverse transcriptase in cDNA synthesis

Reverse transcriptase in cDNA synthesis reverse transcriptase synthesises complementary DNA from mature eukaryotic mRNA transcripts before cloning into genetically engineered host cells.

Plasmid vector insertion and transformation

Plasmid vector insertion and transformation recombinant plasmids carrying marker genes transfer desired DNA sequences into bacterial cells,
- producing genetically modified strains.

Polymerase chain reaction (PCR) cycles

PCR exponentially amplifies DNA through automated thermal cycles: denaturation (95 °C), primer annealing (55-60 °C),
and extension by Taq polymerase (72 °C).

Thermostable Taq DNA polymerase

Taq polymerase isolated from thermophilic bacterium Thermus aquaticus resists thermal denaturation during high-temperature strand separation.

Gel electrophoresis of PCR amplicons

Negatively charged DNA fragments migrate towards the positive anode through an agarose gel matrix at rates inversely proportional to their base pair lengths.

Genome

All the genetic information in an organism.

Restriction endonuclease

Enzyme cutting DNA at a specific recognition sequence.

Marker gene

Gene coding for a detectable substance, used to identify modified cells.

Restriction enzyme

An enzyme that cuts DNA at specific sequences, leaving sticky ends.

Genetically modified

Having had genes from another organism inserted.

Organism

A living thing, such as a plant or an animal.

Plasmids

Convenient vectors

Plasmid

A small ring of DNA in a bacterium carrying extra genes.

Recombinant

Offspring with a new combination of characteristics formed by crossing over.

Electrophoresis

Separation of molecules by size in an electric field

Microarray

Chip with probes used to detect many genes at once.

Protein

A long chain of amino acids containing carbon, hydrogen, oxygen and nitrogen.

Promoter

DNA region where RNA polymerase binds to start transcription.

Key terms

Term	Meaning
PCR	In vitro amplification of DNA by repeated cycles of heating and cooling.
Primer	Short single-stranded DNA that marks where copying starts.
Taq polymerase	Heat-stable DNA polymerase from Thermus aquaticus.
Denaturation	Breaking hydrogen bonds between DNA strands by heating.
Annealing	Binding of primers to complementary single strands.
Elongation	Building of new DNA strands by polymerase.
Thermal cycler	Machine that controls PCR temperatures automatically.

Avoid these mistakes

Mixing up the temperatures — 95 °C denature, 50-60 °C anneal, 72 °C extend.

Saying primers are made by the polymerase — they are supplied in advance.

Forgetting why Taq polymerase is used — it is heat-stable.

Saying PCR copies the whole genome — only the region between the primers is copied.

Last updated: 18 October 2026