Two techniques underpin nearly everything in modern genetics. PCR makes millions of copies of a tiny DNA sample. Gel electrophoresis then sorts the pieces by size. Learn them as a pair, because in practice they are almost always used together.
📘 What you need to know
Gel electrophoresis separates molecules by size (mass) and net charge using an electric current.
DNA is negatively charged because of its phosphate groups, so it moves towards the anode (positive).
Smaller fragments travel further through the gel pores; larger ones stay near the wells.
PCR is the in vitro method of amplifying DNA, producing billions of copies in a few hours.
PCR needs target DNA, primers, Taq polymerase, free nucleotides and a buffer, and runs in a thermal cycler.
Each cycle has three stages — denaturation, annealing, elongation — and doubles the number of molecules.
Gel electrophoresis: the principle
Put a mixture of molecules into a jelly-like gel, apply a voltage across it, and the molecules start to move. How far each one travels in a given time depends on three things.
Factor
Effect on movement
Net charge
Negatively charged molecules move to the anode (positive); positively charged ones move to the cathode (negative)
Size or mass
Small molecules slip through the gel pores quickly; large ones are held back and move slowly
Type of gel
Pore size differs between gels — agarose is used for DNA, polyacrylamide for proteins
For DNA the charge part is easy, because every nucleotide carries a phosphate group and every phosphate group is negative. That means all DNA fragments carry a negative charge, and they all head the same way. So for DNA, separation is effectively by size alone.
This is why DNA gels look so tidy compared with protein gels. With proteins, different molecules carry different charges and can move in different directions, which makes the pattern much harder to read.
Wells go at the cathode end. Every DNA fragment travels the same way; only the distance differs.
📝 Method: separating DNA fragments
Prepare the gel. Set an agarose gel plate in a tank, with a row of small rectangular wells cut into one end.
Submerge it in an electrolyte (buffer) solution that conducts electricity.
Load the samples. Use a micropipette to place DNA fragments into the wells.
Connect the current. The negative electrode goes at the well end, so the negatively charged fragments are pulled away from it towards the anode.
Let it run. Smaller fragments travel faster and end up further from the wells.
Transfer. The fragments are invisible, so they are blotted onto absorbent paper or nitrocellulose, which is heated to separate the two DNA strands.
Visualise. Add probes that bind to the fragments of interest, then develop the pattern.
Two kinds of probe come up in exams:
Radioactive probes, carrying an isotope such as phosphorus-32. They expose X-ray film, producing dark bands.
Fluorescent probes or stains, which glow under ultraviolet light, producing coloured bands.
Sense check for reading a gel: a band close to the wells is a large fragment. A band far from the wells is a small one. Students routinely get this backwards under time pressure.
Polymerase chain reaction (PCR)
Real samples are tiny. A single hair root, a smear of saliva on a cup, a drop of dried blood — there is nowhere near enough DNA to run a useful gel. PCR fixes that by making enormous numbers of copies of one chosen stretch of DNA.
It is described as an in vitro method, meaning it happens in a tube rather than inside a living cell. Starting from as little as a single molecule, PCR can produce billions of copies within a few hours.
Ingredient
What it does
Target DNA
The sample containing the sequence to be copied
Primers
Short sequences that bind either side of the target, marking where copying should start — only the target region is amplified, not the whole genome
Taq polymerase
The enzyme that builds new strands; from the thermophilic bacterium Thermus aquaticus, so it does not denature at high temperature
Free nucleotides
The building blocks of the new strands
Buffer solution
Keeps the pH at the optimum for the enzyme
The Taq detail is a favourite exam question and it has a satisfying answer. A normal human DNA polymerase would be denatured the first time the tube hit 95 °C. Thermus aquaticus lives in hot springs, so its enzyme keeps working.
The three stages of a cycle
A thermal cycler runs the tube through a fixed pattern of temperatures, over and over.
Three temperatures, repeated. Everything else about PCR follows from this pattern.
📝 What happens at each temperature
Denaturation, 95 °C. The heat breaks the hydrogen bonds between the base pairs, separating the double-stranded DNA into single strands.
Annealing, 50–60 °C. Cooling lets the primers bind to the ends of the single strands, marking the region to be copied.
Elongation, 72 °C. This is the optimum for Taq polymerase, which builds complementary strands from free nucleotides, producing new double-stranded molecules.
Notice what is missing. No helicase is needed, because heat does the unzipping instead. That is the one real difference between PCR and replication in a cell.
Why it grows so fast
Every cycle doubles the number of molecules. After n cycles you have 2n times as many as you started with.
Number of copies
copies = starting number × 2n where n is the number of cycles
Slow to start, then startling. Doubling always looks like this.
WORKED EXAMPLE
A PCR reaction starts with a single DNA molecule. Calculate the number of molecules after 25 cycles, and deduce the smallest number of cycles needed to exceed one million molecules. [3]
Step 1: apply the doubling rulecopies = 1 × 225Step 2: evaluate225 = 33 554 432 moleculesStep 3: find when 2n passes one million219 = 524 288 and 220 = 1 048 576, so 20 cycles are neededcheck the boundary rather than guessing — 19 cycles is not quite enough
Putting them together
In practice the order is almost always the same:
Collect a sample and extract the DNA.
Amplify it using PCR, because the original sample is too small to work with.
Cut the amplified DNA with restriction enzymes into fragments.
Separate the fragments by size using gel electrophoresis.
Visualise the bands and compare the pattern.
The next page follows that pipeline through to its real-world uses.
💡 Exam tip
Say DNA is negative because of its phosphate groups. “DNA is negative” alone is often not enough.
Remember the direction: negative DNA moves to the positive anode.
Quote all three PCR temperatures with their stage names. They are commonly asked for directly.
Explain Taq polymerase in terms of not denaturing at high temperature.
Say primers ensure that only the target region, not the whole genome, is copied.
Use 2n for copy-number calculations and show the power before evaluating it.
⚠ Common mix-up
Anode and cathode. In this setup the anode is positive and DNA moves towards it.
Larger fragments do not travel further. They are held back by the gel pores.
PCR does not use helicase. Heat separates the strands instead.
Primers are not the same as probes. Primers start copying in PCR; probes label bands on a gel.
PCR copies a chosen region, not the entire genome.
Restriction enzymes are not part of PCR. They are used afterwards, to cut the amplified DNA.
Up next: Electrophoresis & PCR: Applications — the techniques are done. Now for the interesting part: reading a DNA profile and working out who a sample came from.
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