A single cell’s worth of DNA is far too little to work with, and a mixture of fragments tells you nothing until you sort them. PCR fixes the first problem by copying DNA a billion times over. Gel electrophoresis fixes the second by racing the fragments through jelly. Almost every technique in genetics starts with these two.
📘 What you need to know
Gel electrophoresis separates molecules using an electric current, according to their size or mass and their net charge.
DNA is negatively charged because of its phosphate groups, so it moves towards the anode (positive pole).
Smaller fragments move faster and further through the gel’s pores; larger ones move slowly.
Fragments are invisible, so they are revealed with radioactive labels or fluorescent dyes.
PCR is the in vitro method of DNA amplification, producing huge numbers of copies from tiny samples.
PCR needs: target DNA, primers, Taq polymerase, free nucleotides and a buffer.
Each cycle has three stages: denaturation (95 °C), annealing (50–60 °C) and elongation (72 °C), and each cycle doubles the DNA.
Gel electrophoresis
Gel electrophoresis is used widely in the analysis of DNA, RNA and proteins. The separation happens for three reasons at once.
1. The electrical charge molecules carry
Positively charged molecules move towards the cathode (negative pole); negatively charged molecules move towards the anode (positive pole).
DNA is negatively charged because of its phosphate groups. So in an electric current, DNA always moves towards the anode.
2. The size of the molecules
The gel is full of tiny pores. Smaller molecules move quickly through them; larger molecules move slowly.
So after a fixed time, small fragments have travelled further from the wells than large ones.
3. The type of gel
Different gels have different sized pores, which changes the speed at which molecules move.
Agarose is used for DNA; polyacrylamide is used for proteins.
The charge point is the one students drop. DNA is always negative, so it always runs to the positive electrode — which means the wells must be cut at the negative end of the gel, or the samples run off the wrong way.
The black rectangles on the left are the wells the samples were loaded into. Everything to the right of them is DNA that has migrated.
Preparing DNA for the gel
DNA can be collected from almost anywhere on the body — the root of a hair, saliva from a cup. Before it can be run on a gel and then sequenced or analysed for genetic profiling, it has to be prepared:
First, the number of DNA molecules is increased (amplified) by the Polymerase Chain Reaction.
Then restriction enzymes (DNA-cutting enzymes) chop the DNA into fragments.
🧩 The gel electrophoresis method
Create an agarose gel plate in a tank. Wells — a row of small rectangular holes — are cut into the gel at one end.
Submerge the gel in an electrolyte solution, a salt solution that conducts electricity.
Load the DNA fragments into the wells using a micropipette.
Apply an electrical current. The negative electrode must be at the end with the wells, so the negatively charged phosphates of DNA are pulled towards the anode.
Fragments with a smaller mass move faster and further from the wells than larger ones.
The fragments are not visible, so they are transferred onto absorbent paper or nitrocellulose, which is then heated to separate the two DNA strands.
Add probes to produce a visible result — either a radioactive label such as a phosphorus isotope, which darkens X-ray film into a pattern of dark bands, or a fluorescent stain or dye, which shines under UV light to give coloured bands.
The Polymerase Chain Reaction
PCR is used in most applications of gene technology — DNA profiling, identifying criminals, determining paternity, genetic engineering. During the COVID-19 pandemic it was used in routine diagnostic testing to amplify small amounts of viral RNA.
It is best described as the in vitro method of DNA amplification: it produces large quantities of a specific fragment of DNA or RNA from very small quantities — even a single molecule. Within a few hours, scientists can produce billions of identical copies ready for analysis.
What each reaction needs
Component
What it is for
Target DNA or RNA
The sequence being amplified. The whole genome is not copied — only specific sections that vary between individuals.
Primers
Short sequences that bind to the target section, identifying where copying should start.
DNA polymerase
Builds the new strand. Usually Taq polymerase, from the thermophilic bacterium Thermus aquaticus, because it does not denature at the high temperature of the first stage.
Free nucleotides
The building blocks of the new strands.
Buffer solution
Provides the optimum pH for the reactions.
Why Taq is the clever bit. Every cycle heats the tube to 95 °C, which would denature an ordinary polymerase and mean adding fresh enzyme every single round. Taq comes from a bacterium that lives in hot springs, so it survives the heat and the reaction can be left to run itself.
The three stages of a cycle
PCR runs in a thermal cycler, which automatically provides the optimal temperature for each stage and controls how long is spent at each one.
Notice the temperature never returns to room temperature. The tube simply bounces between three set points for a couple of hours.
🧩 What happens at each temperature
Denaturation — 95 °C. The double-stranded DNA is heated, which breaks the hydrogen bonds holding the two strands together.
Annealing — 50 to 60 °C. The temperature is lowered so the primers can anneal to the ends of the single strands.
Elongation — 72 °C for at least a minute. This is the optimum for Taq polymerase, which builds the complementary strands to produce new identical double-stranded DNA molecules.
Why the numbers get big so fast
Each whole cycle takes a few minutes, and each cycle doubles the DNA. That is the key to the whole technique.
The rule
copies after n cycles = 2n • starting from one molecule
Exponential growth always looks flat before it looks vertical. The last cycle in a run adds more copies than every earlier cycle put together.
Cycles
Copies from one starting molecule
10
210 = 1024
20
220 = 1 048 576, roughly a million
30
230 = 1 073 741 824, just over a billion
Careful with this calculation — several textbooks quote 231 for a 30-cycle run, which is one doubling too many. After n cycles you have 2n copies, so 30 cycles gives 230. Both numbers are “just over a billion”, so the point still stands, but write the exponent that matches the number of cycles you were given.
Worked examples
WORKED EXAMPLE
Explain why DNA fragments move towards the anode during gel electrophoresis, and why smaller fragments end up further from the wells. [3]
Deal with charge firstDNA carries a negative charge because of its phosphate groups.Opposite charges attract, so it is pulled towards the positive anode.Now sizeThe gel contains tiny pores, so smaller fragments pass through more easily and travel further in the same timetwo separate reasons, two separate explanations. Do not merge them.
WORKED EXAMPLE
A PCR run starts with 5 copies of a target sequence and is run for 20 cycles. Calculate the number of copies produced. [2]
Step 1: work out the multiplying factorEach cycle doubles the DNA, so after 20 cycles the amount is multiplied by 220 = 1 048 576Step 2: multiply by the starting number5 × 1 048 576 = 5 242 880About 5.24 × 106 copiesthe starting number is easy to forget. Read the question twice.
WORKED EXAMPLE
Explain why Taq polymerase is used in PCR rather than human DNA polymerase. [3]
Say what happens in the first stageThe mixture is heated to 95 °C to break the hydrogen bonds and separate the strands.What that would do to a normal enzymeA human enzyme would denature at that temperature, so fresh enzyme would be needed every cycle.Now the property of TaqTaq comes from a thermophilic bacterium and is not denatured by the heat, so it works cycle after cyclenaming Thermus aquaticus is a nice extra if you can remember it.
💡 Exam tip
Examiners love asking about the steps of gel electrophoresis. Learn the method in order.
Always give both reasons for separation: size and charge.
Remember anode = positive and cathode = negative, and that DNA runs to the anode.
Learn the three PCR temperatures as a set: 95, 50–60, 72.
For calculations, the factor is 2n where n is the number of cycles — then multiply by the starting amount.
Say in vitro when describing PCR. It is the syllabus wording.
⚠ Common mix-up
Getting the electrodes the wrong way round. Negatively charged DNA moves to the positive anode.
Saying larger fragments travel further. They travel less far, because the pores slow them down.
Thinking the bands are visible on the gel. They are not — probes and dyes make them visible.
Confusing primers with probes. Primers start PCR; probes reveal bands on a gel.
Saying PCR copies the whole genome. It copies only the target section marked by the primers.
Muddling the annealing and extension temperatures. Annealing is the cool step; extension is warmer.
Writing 2n+1 for n cycles. Count the doublings: n cycles means n doublings.
Up next: Electrophoresis & PCR: Applications — putting both techniques to work on paternity tests and crime scenes.
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