The big idea: Often a scientist starts with far too little DNA to study — a single hair, a drop of blood, or a trace of cells.
How PCR solves it
- PCR (the polymerase chain reaction) makes millions of copies of a chosen piece of DNA
- It runs automatically, inside a machine called a thermal cycler
- It works by repeating a three-step cycle over and over
- The amount of DNA roughly doubles every cycle, so after about 30 cycles a tiny sample becomes enough to analyse
- PCR (polymerase chain reaction)
- A laboratory technique that makes many copies of (amplifies) a specific length of DNA.
- Amplify
- To make many copies of a piece of DNA, so a tiny sample becomes a large, usable amount.
- Thermal cycler
- The machine that runs PCR by repeatedly heating and cooling the sample through the cycle's temperatures.
- Primer
- A short single strand of DNA that binds to a matching sequence and marks where copying should begin.
- Taq polymerase
- The heat-stable enzyme used in PCR; it adds DNA nucleotides to build the new complementary strand.
Why PCR is so powerful: Because the DNA roughly doubles each cycle, the increase is exponential: 1 → 2 → 4 → 8 → 16 …
Just 30 cycles turns one copy into over a billion — which is why PCR can work from a vanishingly small starting sample.
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Each PCR cycle has three steps, and the key to the whole technique is that the temperature is changed at each step for a specific reason.
Why it is asked: Get the temperatures the wrong way round and PCR fails, so questions often ask you to explain why each temperature is needed.
One cycle = three temperatures
Denaturation (~95 °C)
Very high heat breaks the hydrogen bonds holding the two strands together, so the double helix separates into two single strands.
Annealing (~55 °C)
Cooling lets short primers bind (anneal) to their matching sequence on each single strand, marking the start point for copying.
Extension (~72 °C)
At Taq polymerase's optimum temperature, the enzyme adds nucleotides to each primer, building a new complementary strand.
| Step of one PCR cycle | Temperature (approx.) | What happens — and why that temperature |
|---|---|---|
| 1. Denaturation | ~95 °C (very hot) | The high heat breaks the hydrogen bonds, separating the double helix into two single strands so each can be copied. |
| 2. Annealing | ~55 °C (cooler) | Cooling lets short primers bind (anneal) to their matching sequence on each single strand, marking where copying starts. |
| 3. Extension (elongation) | ~72 °C (warm) | This is the optimum temperature for Taq polymerase, which adds free DNA nucleotides to the primer to build a new complementary strand. |
Why Taq polymerase — not an ordinary one: The denaturation step reaches about 95 °C. An ordinary enzyme would be denatured (its shape destroyed) at that temperature and would stop working.
Taq is heat-stable: Taq polymerase is heat-stable (thermostable) — it survives the high heat without denaturing, so the same enzyme keeps working every cycle and does not need to be replaced.
Taq comes from Thermus aquaticus, a bacterium that lives in hot springs, so its enzymes naturally tolerate high temperatures.
| Feature of Taq polymerase | Why it matters in PCR |
|---|---|
| It is heat-stable (thermostable) | It is not denatured by the ~95 °C denaturation step, so the same enzyme keeps working cycle after cycle. |
| It comes from a heat-loving bacterium | Taq is from Thermus aquaticus, which lives in hot springs, so its enzymes naturally tolerate high temperatures. |
| Its optimum is ~72 °C | It works fastest at the warm extension step, exactly where new strands are built. |
| A normal (human) polymerase would NOT work | An ordinary DNA polymerase would be denatured at 95 °C and would have to be replaced every cycle. |
Once PCR has made plenty of DNA, scientists often want to see and compare the fragments. They do this with gel electrophoresis, which sorts fragments by size.
Gel electrophoresis — sorting by size
Load and switch on
DNA samples are loaded into wells at one end of a jelly-like gel, and an electric current is switched on.
DNA moves
DNA carries a negative charge, so it is pulled through the gel towards the positive electrode.
The gel sieves
The gel acts like a sieve: smaller fragments move further, while larger fragments are held back near the wells.
Bands
The result is a pattern of bands sorted by size.
| Feature of gel electrophoresis | What it does |
|---|---|
| DNA fragments are loaded into wells | Each well (lane) holds one sample's mixture of DNA fragments. |
| An electric field is applied | DNA is negatively charged, so it is pulled through the gel towards the positive electrode. |
| The gel acts as a sieve | Smaller fragments slip through the gel mesh easily; larger fragments are held back. |
| Fragments separate by size | Smaller fragments travel further; larger fragments stay nearer the wells — forming a pattern of bands. |
PCR
- Makes copies of a DNA piece (amplifies it)
- Repeats a three-step thermal cycle
- Needs heat-stable Taq polymerase + primers
- DNA roughly doubles each cycle
Gel electrophoresis
- Separates DNA fragments to compare them
- Uses an electric field across a gel
- Sorts by size (smaller travels further)
- Produces a pattern of bands
An easy way to remember: PCR copies; the gel sorts.
Temperatures in order: Hot splits (95 °C denatures), cool sticks (55 °C anneals primers), warm builds (72 °C Taq extends).
On a gel: small = fast = far.
Practice with exam-style questions
Answer exam-style questions and get AI feedback that shows you exactly what examiners want to see in a full-marks response.
The big idea: Questions often show a temperature graph of PCR or a gel. Each one is read the same way every time.
The points to remember
- On a PCR graph: the highest step (about 95 °C) separates the strands.
- The lowest step (about 55 °C) lets primers bind; the middle step (about 72 °C) is where Taq builds.
- On a gel: DNA is negative, so every fragment moves towards the positive electrode.
- Smaller fragments travel further; the band nearest the wells is the longest fragment.
- A lane with no template DNA shows no band of copied DNA; fewer cycles give fainter bands.
Remember it as: Hot splits, cool sticks, warm builds. Small runs far.
Real example: the first PCR. Kary Mullis worked out PCR in 1983 and shared the 1993 Nobel Prize in Chemistry for it. Today a thermal cycler runs 30 cycles in about two hours.
Two traps: DNA never moves towards the negative electrode: it is negatively charged.
A faint band means less DNA, not smaller fragments: size is shown by how far a band travelled.
How this skill is assessed: Data questions often show a PCR gel. Each part rewards reading the bands carefully.
| Question | What to give |
|---|---|
| Explain why the temperature is changed at each step of a cycle | A separate reason for the hot, cool and warm steps |
| A reason Taq polymerase is suitable for PCR (1 mark) | It is heat-stable and is not denatured at ~95 °C |
| State the no-DNA control | The lane with no band |
| Predict how the gel would change | Read the bands carefully |
Exam-style question — explain the PCR temperatures
Explain why the temperature is changed at each of the three steps of a PCR cycle. [3]
How to score all three marks
- Denaturation (high temperature). Heating to about 95 °C breaks the hydrogen bonds, separating the double helix into two single strands so each can act as a template.
- Annealing (lower temperature). Cooling to about 55 °C lets the primers bind (anneal) to their complementary sequences on the single strands, marking where copying begins.
- Extension (intermediate temperature). About 72 °C is the optimum for Taq polymerase, which adds nucleotides to the primer to build a new complementary strand.
Final answer
High heat (~95 °C) separates the strands by breaking hydrogen bonds; a cooler step (~55 °C) lets primers anneal; and ~72 °C is Taq's optimum so it can extend the new strand.
Why this scores full marks: Each step is given a distinct reason tied to its temperature — strands separating, primers binding, Taq extending.
A 3-mark 'explain' wants the cause linked to the effect at each step, not just the temperatures listed.
| Step of one PCR cycle | Temperature (approx.) | What happens — and why that temperature |
|---|---|---|
| 1. Denaturation | ~95 °C (very hot) | The high heat breaks the hydrogen bonds, separating the double helix into two single strands so each can be copied. |
| 2. Annealing | ~55 °C (cooler) | Cooling lets short primers bind (anneal) to their matching sequence on each single strand, marking where copying starts. |
| 3. Extension (elongation) | ~72 °C (warm) | This is the optimum temperature for Taq polymerase, which adds free DNA nucleotides to the primer to build a new complementary strand. |