A single hair or a speck of blood holds too little DNA to study. The polymerase chain reaction copies one chosen section over and over, and gel electrophoresis then sorts the copies by length so they can be compared.
The polymerase chain reaction (PCR) clones and amplifies a chosen section of DNA. A tube holds the template DNA, two primers, free DNA nucleotides, Taq polymerase and buffer, and a thermal cycler repeats three steps: denaturation at about 95 °C (hydrogen bonds break, strands separate), annealing at about 55 °C (primers bind to complementary sequences at each end of the target) and extension at about 72 °C (Taq polymerase adds nucleotides to the primers). Every cycle doubles the DNA, so n cycles give 2n copies. Taq polymerase comes from a bacterium of hot springs and is thermostable, so it survives 95 °C. In gel electrophoresis, DNA is loaded into wells in an agarose gel; its phosphate groups make it negatively charged, so it moves towards the anode, and shorter fragments move further through the mesh of the gel.
Fig. 1: two cycles of PCR in a thermal cycler; each cycle of denaturation, annealing and extension doubles the number of copies of the target DNA
Section 01
The polymerase chain reaction.
Press Run 10 cycles. Watch the STAGE box change from denaturation (95 °C) to annealing (55 °C) to extension (72 °C), the red temperature trace build up on the graph, and the COPIES box double at the end of each cycle until it reaches 1,024.
What goes in the tube: the DNA to be copied (template), two different primers, free DNA nucleotides (with A, T, C and G), Taq polymerase and a buffer that keeps the pH and ion concentrations suitable for the enzyme.
Denaturation, about 95 °C: the hydrogen bonds between complementary bases break, so the double helix separates into two single strands. No enzyme is needed for this.
Annealing, about 55 °C: cooling lets the primers bind by complementary base pairing to the start of the target sequence on each strand. Primers are short, single-stranded pieces of DNA (about 20 bases). They are needed because DNA polymerase can only add nucleotides to an existing strand, and their sequence decides exactly which section of DNA is copied.
Extension, about 72 °C: Taq polymerase binds and adds free nucleotides to the end of each primer, building a strand complementary to the template. 72 °C is the optimum temperature of Taq polymerase.
Doubling: each new strand is a template in the next cycle, so the number of copies doubles each cycle: 2n after n cycles from one molecule, which is over a billion after 30 cycles. A cycle takes a few minutes, so millions of copies are made in a couple of hours.
Taq polymerase comes from Thermus aquaticus, a bacterium that lives in hot springs. Its enzymes are thermostable, so they are not denatured during the 95 °C steps and do not need replacing every cycle; human DNA polymerase would be denatured.
Uses: amplifying DNA from tiny samples for forensic DNA profiling, detecting pathogens or disease alleles, and making many copies of a gene for genetic engineering.
Examiner trap
Primers are DNA, not RNA, in PCR, and there are two of them: one binds to each strand at opposite ends of the target, so the section between them is what gets copied.
Section 02
Sorting DNA by length.
Press Switch on the current. The bands start in the wells at the negative cathode and move towards the positive anode. Compare the crime scene lane (900, 450 and 250 base pairs) with suspect A and suspect B, using the ladder of known sizes (1000 down to 100 base pairs).
Preparing the samples: DNA is cut into fragments with restriction endonucleases, or a chosen region is amplified by PCR. The samples are loaded into wells at one end of an agarose gel covered in buffer.
Why DNA moves: each nucleotide has a phosphate group, so DNA is negatively charged. When a voltage is applied across the gel, the fragments move away from the negative electrode (cathode) towards the positive electrode (anode).
Why fragments separate: the gel is a mesh of fibres that acts like a sieve. Short fragments pass through it more easily and travel further in a given time; long fragments are held back and stay closer to the wells. DNA has the same charge per unit length, so separation is by length alone.
Seeing the bands: the DNA is stained, or labelled with a fluorescent dye or a radioactive probe, so each group of fragments of the same length shows as a band.
The ladder: a lane of fragments of known lengths run on the same gel lets the length of unknown fragments be estimated by comparing how far they moved.
DNA profiling: regions of the genome with variable numbers of short repeated sequences differ in length between people. Two samples whose bands all match very probably came from the same person; in the simulator suspect B matches the crime scene sample and suspect A does not.
Examiner trap
Distance travelled is not proportional to length: it falls roughly with the logarithm of the fragment length, so always estimate sizes by comparing with a ladder on the same gel.
Stage 1 · Learn
Check what the sim just showed you
Four quick checks. Each correct answer earns XP and records this skill on your profile.
Quick check+10 XP
You press Run 10 cycles in the PCR simulator, starting with one DNA molecule. What does the COPIES box show at the end?
Quick check+10 XP
During the annealing stage shown in the simulator, what happens to the yellow primers?
Quick check+10 XP
In the gel simulator, which sample matches the crime scene DNA?
Quick check+10 XP
Which crime scene band travels furthest from the wells in the simulator, and why?
Stage 2 · Exam
Exam-style questions
Unlocks once the four checks above are done. Worth more XP, written to A Level Paper 4 standard.
Finish the four checks above to unlock the exam questions
Exam style+20 XP
Why is Taq polymerase used in PCR rather than DNA polymerase from human cells?
Exam style+20 XP
Twenty molecules of target DNA are amplified by PCR for 15 cycles. Assuming every molecule is copied in every cycle, how many molecules are there at the end?
Exam style+20 XP
Why do DNA fragments move towards the anode during gel electrophoresis?
Exam style+20 XP
What is the function of the primers in PCR?
Skill unlocked
PCR and gel electrophoresis, mastered.
This skill is now recorded on your profile. Keep the chain going.