The shape of the lesson
By the end of the lesson, learners can
- The polymerase chain reaction: Press Run 10 cycles.
- Sorting DNA by length: Press Switch on the current.
- 95 °C separate, 55 °C primers bind, 72 °C Taq extends.
- Copies = starting number × 2n after n cycles.
- DNA is negative, so it runs to the anode.
- Shorter fragments travel further; compare with a ladder.
One picture every idea returns to
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.
Forty-five minutes, phase by phase
| Time | Phase | What happens in the room | Grouping |
|---|---|---|---|
| 0 to 5 min | Hook: predict | Open with the question in the title, Copy it a billion times, then sort it by size.. A single hair or a speck of blood holds too little DNA to study. Put the figure on the board: it is the one picture every idea in the lesson returns to. | Whole class, then pairs |
| 5 to 17 min | Teach and check: The polymerase chain reaction | Press Run 10 cycles. Project tab 3 of the lab (Genetic technology lab, PCR) and let a student drive it while the class predicts. | Whole class, then pairs |
| 17 to 30 min | Teach and check: Sorting DNA by length | Press Switch on the current. Project tab 4 of the lab (Genetic technology lab, gel electrophoresis) and let a student drive it while the class predicts. | Think-Pair-Share |
| 30 to 36 min | Check | The four Stage 1 questions from the student lesson, answered on mini whiteboards or by show of fingers. Re-teach the one most get wrong. | Individual, then whole class |
| 36 to 43 min | Plenary | The exam-style questions from Stage 2, one at a time: thirty seconds alone, then a random call. The feedback lines in the key give the examiner's wording. | Individual, random call |
| 43 to 45 min | Exit and homework | Set the worksheet below (6 questions, 18 marks). Learners self-assess against the objectives before they leave. | Individual |
Protect the parts that carry the learning
The sequence carries about fifty minutes of material, so the lab is driven by one student while the class predicts rather than explored freely. Times are a guide.
Protected: the check at 30 minutes. It is the only individual read on whether the key idea landed, and it is not cut.
Cut first if the build overruns: the last check in Sorting DNA by length, never the check or the plenary.
In a 60 minute block: run worksheet questions 1 to 3 in class as a Rally Coach set and model both the hardest worked example and its check live.
Think-Pair-Share
Pose the question, give thirty seconds of silent thinking, then a minute to compare with a partner before a few pairs share with the class. The silent minute is the part that cannot be cut.
The full structure, with its variations and the other twelve, is on the cooperative-learning page.
What to head off, and how
| Trap learners fall into | Teaching move that pre-empts it |
|---|---|
| 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. | Return to The polymerase chain reaction: ask a pair to state the correct version and show it on the lab before moving on. |
| 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. | Return to Sorting DNA by length: ask a pair to state the correct version and show it on the lab before moving on. |
PCR and gel electrophoresis: worksheet
6 questions, 18 marks. Written in the style of the papers; no past paper question is reproduced.
Describe what happens during each of the three stages of one cycle of the polymerase chain reaction.
Explain why Taq polymerase is used in PCR and why the extension stage is carried out at 72 °C.
A forensic sample contains 50 copies of a target DNA sequence. Calculate how many copies there will be after 25 cycles of PCR, assuming every molecule is copied in every cycle. Give your answer in standard form.
Explain how gel electrophoresis separates DNA fragments of different lengths.
On a gel, ladder fragments of 1000, 500 and 250 base pairs travelled 12 mm, 22 mm and 32 mm from the wells. An unknown fragment travelled 26 mm. Estimate its length and explain your reasoning.
DNA from a blood stain at a crime scene and from three suspects was amplified by PCR and run on a gel. The blood stain gave bands at 900, 450 and 250 base pairs. Suspect A gave 800, 450 and 150; suspect B gave 900, 450 and 250; suspect C gave 900, 600 and 250. State and explain which suspect the evidence links to the blood stain, and why PCR was needed.
Answer key and marking notes
- Denaturation (about 95 °C): hydrogen bonds between complementary bases break and the two DNA strands separate.
- Annealing (about 55 °C): primers bind by complementary base pairing to the ends of the target sequence on each single strand.
- Extension (about 72 °C): Taq polymerase adds free DNA nucleotides to the primers, forming new complementary strands.
- Taq polymerase comes from Thermus aquaticus, a bacterium living in hot springs, so it is thermostable.
- It is not denatured at 95 °C during denaturation, so it does not need to be added again every cycle (a human enzyme would be denatured).
- 72 °C is close to its optimum temperature, so it adds nucleotides quickly; this temperature is also high enough to stop primers binding to the wrong sequences.
- Number of copies = 50 × 225 = 50 × 33 554 432.
- = 1 677 721 600 = 1.68 × 109 copies.
- DNA samples are placed in wells at the cathode (negative) end of an agarose gel in a buffer, and a voltage is applied.
- DNA is negatively charged because of its phosphate groups, so the fragments move towards the anode (positive electrode).
- The gel acts as a mesh or sieve; shorter fragments move through it more easily and so travel further in the same time.
- Fragments of the same length form a band, made visible by a stain or fluorescent / radioactive label; lengths are estimated by comparison with a ladder of known lengths.
- It travelled further than the 500 base pair fragment but not as far as the 250 base pair fragment, so its length is between 250 and 500 base pairs.
- Each halving of length adds 10 mm, so distance is proportional to the logarithm of length, not to length itself.
- 26 mm is 4/10 of the way from 22 to 32 mm, giving about 380 base pairs (accept 350 to 400).
- Suspect B: all three bands match the blood stain in length (position on the gel).
- Suspects A and C share only some bands, so the stain could not have come from them.
- The stain contained very little DNA, so PCR was used to amplify the regions to give enough DNA to see on the gel.
8 multiple-choice questions on this lesson
From the topic question bank. Assign them in the app for a marked score in your gradebook, or run the topic as a class game.
Which list contains everything that must be placed in the tube for a polymerase chain reaction?
- Atemplate DNA, two primers, free DNA nucleotides, Taq polymerase and buffer
- Btemplate DNA, one primer, free RNA nucleotides, RNA polymerase and buffer
- CmRNA, reverse transcriptase, free DNA nucleotides and DNA ligase
- Dtemplate DNA, a restriction endonuclease, DNA ligase and plasmids
Why must the temperature be lowered from about 95 °C to about 55 °C for annealing in PCR?
- Aat 95 °C the hydrogen bonds holding a primer to the template would break, so primers cannot stay bound
- BTaq polymerase is denatured at any temperature above 55 °C
- Cprimers are broken down into nucleotides at temperatures above 55 °C
- Dthe two template strands must rejoin before the primers can bind
Starting from a single molecule of target DNA, what is the minimum number of PCR cycles needed to produce at least one million copies, assuming the DNA doubles in every cycle?
- A20
- B10
- C6
- D1000
The gel shows DNA profiles of a mother, her child and two men. Every band in a child's profile must come from one of the parents. Which conclusion is supported?
- Aman 2 could be the father, because he has the child's band that did not come from the mother
- Bman 1 is the father, because he shares no bands with the mother
- Cneither man could be the father, because neither has two bands matching the child
- Dthe mother is not the child's mother, because she shares only one band with the child
At which end of the gel are the wells placed in gel electrophoresis of DNA, and why?
- Aat the cathode end, because negatively charged DNA moves away from it towards the anode
- Bat the anode end, because DNA is attracted towards the negative cathode
- Cat the anode end, because shorter fragments need the longest possible path
- Dat either end, because DNA has no charge and spreads out by diffusion
In practice, after 30 cycles of PCR there are far fewer than 230 copies of the target DNA. Which is the most likely reason?
- Aprimers and free nucleotides are used up in later cycles, so the DNA no longer doubles each cycle
- BTaq polymerase is denatured during the first denaturation step
- Cthe products of earlier cycles cannot be used as templates in later cycles
- Deach cycle produces shorter DNA, which eventually becomes too short to copy
On a gel, a 1000 base pair ladder fragment moved 10 mm and a 100 base pair fragment moved 40 mm. Distance moved is proportional to the logarithm of fragment length over this range. An unknown fragment moved 25 mm. What is its approximate length?
- A320 base pairs
- B550 base pairs
- C250 base pairs
- D400 base pairs
To test for a viral infection, primers are designed that are complementary only to sequences in the virus genome. DNA from a patient's sample is amplified by PCR and run on a gel. Why does a band of the expected size appear only if the virus is present?
- Athe primers can only anneal to viral sequences, so only viral DNA is amplified enough to be seen
- BTaq polymerase can only copy DNA that comes from a virus
- Chuman DNA is destroyed at 95 °C, but viral DNA is not
- Dviral DNA moves through the gel, but human DNA stays in the well
Quiz key
PCR needs the DNA to copy, a primer for each strand, DNA nucleotides to build new strands, a thermostable DNA polymerase and a buffer. One primer would copy only one strand; RNA nucleotides and RNA polymerase make RNA, not DNA. Reverse transcriptase and mRNA are used to make cDNA. Restriction enzymes, ligase and plasmids are tools for making recombinant DNA, not for amplifying it.
Primers bind by complementary base pairing, held by hydrogen bonds. At 95 °C these bonds break, which is why the strands separate; primers can only anneal once the mixture is cooled. Taq polymerase is thermostable and works best at about 72 °C. Heat breaks hydrogen bonds, not the phosphodiester bonds within a primer. If the template strands rejoined, the primers could not bind at all.
219 = 524 288, which is not enough, and 220 = 1 048 576, so 20 cycles are needed. 10 cycles give only 210 = 1024. 6 comes from treating one million as 106 and forgetting that copies double, not multiply by ten. 1000 cycles confuses the number of cycles with a number of copies.
The child's upper band matches the mother. The child's second band is not in the mother's lane, so it came from the father; only man 2 has a band at that position, so he could be the father. Sharing no bands with the mother says nothing about paternity. A child inherits about half its bands from each parent, so the father needs to match only the paternal band. Sharing one of two bands with the mother is exactly what is expected.
The phosphate groups give DNA a negative charge, so it is repelled by the cathode and attracted to the anode; the wells go at the cathode end so the DNA can travel the length of the gel. DNA is not attracted to the cathode. Where the wells go depends on charge, not on fragment length. DNA is charged and moves because of the electric field, not by diffusion.
Every new strand uses a primer and nucleotides. As the DNA builds up, these run low (and the enzyme becomes limiting), so the efficiency of each cycle falls and the curve levels off. Taq is thermostable, so it survives denaturation. New strands are templates in later cycles; that is why copying is exponential at first. Copies settle at the length between the two primers; they do not keep getting shorter.
25 mm is halfway between 10 and 40 mm, so log10(length) is halfway between 3.0 and 2.0, that is 2.5. Length = 102.5 ≈ 316, about 320 base pairs. 550 is the arithmetic mean of 1000 and 100, which assumes distance is proportional to length itself. 250 base pairs would need a distance closer to the 100 base pair band (about 28 mm), and 400 base pairs a distance of about 22 mm, so neither fits 25 mm.
Amplification needs both primers to anneal on either side of a target. If the viral sequence is absent, the primers have nowhere to bind, nothing is amplified, and no band appears. Taq polymerase copies any template once a primer is bound. 95 °C separates strands but does not destroy human DNA. All DNA is negatively charged and moves through the gel; human DNA is simply not amplified to a visible amount.
What to project
The student lesson embeds these tabs. Open them full screen on the projector; every slider has a drag handle on the stage.
Slides for this lesson
A projectable deck for every pack is on the way. Until then, present the student lesson full screen: its sections, figure and lab tabs follow the same order as the plan above.