What you need to know
- 6.17DNADeoxyribonucleic acid: a double-stranded polynucleotide that carries the genetic code. is amplified by the polymerase chain reactionA method that makes many copies of a DNA sequence by repeated cycles of heating (denaturing), cooling (annealing primers) and extending with Taq polymerase..
Gel separation is the next lesson. A theoretical 2^n assumes every molecule is copied every cycle. Taq is used because it survives the hot step. You do not need the amino-acid sequence of the enzyme.
By the end, you should be able to…
- Name the ingredients and the three temperatures of one cycle.
- Explain why the primers decide which region is copied.
- Calculate a theoretical copy number and state the efficiency assumption.
Build your understanding
What goes into the tube
polymerase chain reactionA method that makes many copies of a DNA sequence by repeated cycles of heating (denaturing), cooling (annealing primers) and extending with Taq polymerase. makes many copies of a chosen region of DNADeoxyribonucleic acid: a double-stranded polynucleotide that carries the genetic code.. The tube needs the template DNA, two primers, a supply of nucleotideThe monomer of nucleic acids: a pentose sugar, a phosphate group and a nitrogenous base., a buffer, and a heat-stable DNA polymeraseThe enzyme that joins free DNA nucleotides into a new strand, forming phosphodiester bonds, during replication.. The usual school enzymeA globular protein that acts as a biological catalyst, speeding up a specific reaction. is Taq polymeraseA heat-stable DNA polymerase from a thermophilic bacterium, used in PCR., from a bacterium that lives at high temperature, so the enzyme is not destroyed when the tube is heated to separate the strands.
Primers are short single strands that base-pair with the two ends of the region you want. They are why PCR is specific. Without primers that match the target, the polymerase has nowhere to start. You do not copy the whole genome unless the primers are chosen to do something that broad.
Three steps, then repeat
denaturationA permanent change to a protein’s tertiary structure, for example by high temperature or extreme pH, so an enzyme’s active site no longer fits its substrate., about 95 °C, separates the double helixThe shape of DNA: two antiparallel polynucleotide strands joined by base pairs and twisted into a spiral.. hydrogen bondA weak attraction between a partially positive hydrogen atom and a partially negative atom such as oxygen or nitrogen. break. The sugar-phosphate backbones stay intact. Annealing, often about 50 to 65 °C depending on the primers, lets the primers bind. Extension, about 72 °C, is where Taq adds nucleotides to the primer, building a complementary strand. Then the cycle returns to 95 °C.
Three steps, then repeat (continued)
Heat is doing the strand separation that helicaseThe enzyme that unwinds DNA and breaks the hydrogen bonds between bases, separating the two strands. would do in a cell. Each new strand can be a template in the next cycle. If you start with one target molecule and every copy works, the number of target molecules is 2 to the power n after n cycles. One molecule and 10 cycles gives 1024. Thirty cycles is about a billion-fold. Real reactions are less than perfect, so these are theoretical maxima. State that assumption.
Follow the process
About 95 °C denatures the double helix, so hydrogen bonds break and the strands separate → about 50–65 °C lets the two primers anneal to the ends of the chosen region → about 72 °C lets Taq polymerase extend from each primer → the cycle returns to about 95 °C, and each new strand can be a template. Theoretical copies = starting number × 2ⁿ only if every molecule is copied every cycle. The primers choose the region. Taq is used because it survives the hot step. Helicase is not in the tube.
What a bright tube does not mean
PCR amplifies whatever matches the primers, including a contaminating cell from the operator. A negative control with no template should stay blank. A positive control should amplify. Copies are not a profile until they are separated and compared. That is electrophoresis.
The invention of PCR is recognised by the 1993 Nobel Prize in Chemistry to Kary Mullis. The same prize also recognised Michael Smith’s work on site-directed mutagenesis. You need the method, not the biography.
NOT: PCR does not copy the whole genome at body temperature, and helicase is not in the tube.
- The tube needs template, two primers, nucleotides, buffer and Taq
- Taq survives the 95 °C step; an ordinary polymerase would not
- One target and 10 perfect cycles give 2¹⁰ = 1024 copies
- Thirty perfect cycles are about a billion-fold, a theoretical maximum
- A no-template control should stay blank, because contamination is also copied
Two traps this diagram blocks
- Do not put helicase in the PCR tube.
- Do not say one cycle copies the whole genome once at 37 °C.
From the diagram alone
Question
Explain the three temperature steps of one PCR cycle and calculate the theoretical number of molecules after 10 cycles, starting from one target molecule, if every copy works. [4]
Show a model answer
About 95 °C separates the strands, about 50–65 °C lets the primers bind, and about 72 °C lets Taq polymerase extend them. Each cycle can double the number, so 10 cycles give 2¹⁰ = 1024 molecules. That figure assumes every molecule is copied every cycle.
Make your reasoning visible
Build the explanation
X happens because Y, leading to Z. X: only the chosen region is copied. Y: the primers base-pair with its two ends, so Taq has a place to start. Z: after n cycles the theoretical number of target molecules is the starting number times 2ⁿ, if every copy works. Incomplete: “polymerase chain reactionA method that makes many copies of a DNA sequence by repeated cycles of heating (denaturing), cooling (annealing primers) and extending with Taq polymerase. copies all the DNADeoxyribonucleic acid: a double-stranded polynucleotide that carries the genetic code. at body temperature using helicaseThe enzyme that unwinds DNA and breaks the hydrogen bonds between bases, separating the two strands..” Improved: “Heat at about 95 °C separates the strands, primers select the region, and Taq extends at about 72 °C.”
Primers, then polymerase
If the question says “how is the region chosen”, the answer is the primers, not Taq.
Write the power
Theoretical copies = starting number × 2^n. Say “if efficiency is 100 percent”.
Key explanations
- Real reactions are less efficient, so 1024 is a theoretical maximum. A negative control with no template should produce no product.
Avoid this misconception
The claim: PCR copies the whole genome once, at body temperature, using helicase.
The correction: PCR copies the region between two primers, by repeating about 95, then about 50–65, then about 72 °C. Heat separates the strands. Taq polymeraseA heat-stable DNA polymerase from a thermophilic bacterium, used in PCR. builds the new strands.
How do you turn one molecule of DNA into a billion copies?
A crime scene or a seed may leave only a trace of DNA. PCR copies one chosen region over and over, doubling it each cycle, until there is enough to see.
What the model shows
One cycle of the polymerase chain reaction at three temperatures, and the number of target molecules after many cycles, in theory and in a real tube.
What to look for
What happens at 95, 55 and 72 °C. How the primers pick the region. How fast the number grows, and what a negative control should show.
What it tells us
Theoretical copies = starting number × 2ⁿ after n cycles, if every molecule is copied every cycle. Primers make PCR specific, and Taq survives the hot step.
Be careful what you claim
Copy numbers are model values, and the real-tube curve is a simple model. Gel separation is the next lesson (choose Gel under Part).
Try it in the model: press Next step four times and watch one DNA molecule become two.
The polymerase chain reaction
PCR copies a chosen region of DNA, doubling it each cycle in theory. A gel then sorts the copies by length beside a size ladder. All copy numbers are model values and all samples are fictional.
What you see
- PCR: a thermocycler trace for one cycle (95, 55 and 72 °C) and one DNA molecule going through it: the strands separate, the primers bind and Taq builds new strands.
- A log graph of target copies against cycle number: the theoretical 2ⁿ line and a real tube that levels off.
- Gel: a size ladder and four fictional samples. The loading dye moves while the gel runs; the bands appear when you stain it. A standard curve of distance against length sits beside it.
How to use it
- Under Part, choose PCR or Gel.
- PCR: Next step moves one stage at a time. Run 1 cycle animates a cycle. Fast-forward runs 5 or to 30 cycles. Change the starting molecules and the sample.
- Gel: set the voltage and press Run the gel, then Stain the gel. Click a band to estimate its length. Change the number of loci and the sample set.
What it shows
- DNA amplified by the polymerase chain reaction (6.17): the three temperatures, primers, Taq and the 2ⁿ calculation.
- Gel electrophoresis separating DNA fragments by length (6.18).
- DNA profiling to identify individuals and show relationships, in people, plants and animals (6.19).
Limits of the model
- Copy numbers are model values. The real tube is a simple model of efficiency falling as reagents run out.
- Strictly, fragments of exactly the target length first appear in cycle 3, so the exact count is 2ⁿ − 2n from one molecule. The exam uses starting number × 2ⁿ, as this model does.
- All band sizes are fictional and there are only two loci. Real profiles use many loci, and a match is evidence, not a probability or proof of guilt.
- Distance is modelled as falling with log(length) over the gel’s working range; real gels depend on the agarose percentage and buffer.
Guided tour · step 1 of 10
- In Thermocycler, just below these steps, press Next step.
- The thermocycler heats the tube to about 95 °C.
Heat breaks the hydrogen bonds between the bases, so the two strands separate. The sugar-phosphate backbones stay intact. No helicase is needed.
Thermocycler
Theory: 1 × 2⁰ = 1 target molecules after 0 cycles, if every molecule is copied every cycle.
Questions
Check your understanding
Try each question before opening the suggested answer.
Check your understanding (continued)
Practice question
Retrieval and explanation · suggested answer
State the three temperature steps of one PCR cycle and what each does.
Show suggested answer
About 95 °C separates the strands. About 50–65 °C lets primers bind. About 72 °C lets Taq polymerase extend the new strands.
Practice question
Retrieval and explanation · suggested answer
Why is Taq polymerase used rather than a human DNA polymerase?
Show suggested answer
Taq is not destroyed by the 95 °C step. A typical human polymerase would be denatured.
Practice question
Retrieval and explanation · suggested answer
Theoretical calculation: 4 target molecules, 5 cycles, 100 percent efficiency. How many target molecules?
Show suggested answer
4 × 2^5 = 4 × 32 = 128.
Practice question
Retrieval and explanation · suggested answer
What is the job of the primers?
Show suggested answer
They bind the two ends of the chosen region and give the polymerase a place to start, so only that region is amplified.
Take the idea further
Apply the same specification knowledge to an unfamiliar problem. Any extra assumptions are supplied in the question.
Stretch question
A tube was meant to amplify a plant gene. The negative control, with no plant DNA added, also produced a product of the same length. Give one explanation. What does that do to a claim that the plant sample contained the gene? Assume the primers and the length are as intended.
Show a hint
Copies can come from DNA you did not mean to add.
Explore the reasoning
The primers found matching DNA in the control, often contamination from a previous reaction or from the operator. You cannot claim the plant sample is the source of the product, because the same product appeared without it. The run needs to be repeated with cleaner technique. Same length means the contamination matched the target, not that the gel failed.
Recall this lesson
Lesson recall questions
Question 1 of 5: How does denaturation allow primers to bind in the next stage?
PCR amplification
Practice question
Retrieval and explanation · suggested answer
How does denaturation allow primers to bind in the next stage?
Lesson: 6C 4: Polymerase Chain Reaction (PCR)
Try this from memory first.
Put your biology to work
Explore where these ideas appear in the world. Enrichment, not extra required recall.
PCR is amplification, not a result by itself
6C · Polymerase Chain Reaction (PCR)Yourgenome describes PCR as a way to make millions of copies of a small region of DNA, using template, primers, nucleotides and Taq, through repeated heating and cooling.…Specification 6.17Read & use the scienceClose article
Why this matters
Yourgenome describes PCR as a way to make millions of copies of a small region of DNA, using template, primers, nucleotides and Taq, through repeated heating and cooling. It also notes uses such as detecting a pathogen. A positive amplification still has to be interpreted. It is not, on its own, a DNA profile or a diagnosis. Primers determine which region is copied. Negative and positive controls are essential when an assay is sensitive enough to amplify tiny amounts of contaminating DNA.
Back to the specification
Pearson Edexcel IAL Biology · Issue 2, February 2021 · Unit 4, pp. 29–32. The application extends beyond required recall.
- 6.17 know how DNA can be amplified using the polymerase chain reaction (PCR)
Use the science
Why is “the tube went positive, so the person is guilty” larger than both the page and the specification?
Explore a possible response
PCR only shows that a sequence matching the primers was copied. Contamination can do that. Identity and relationships need a profile of several regions, which is the next lesson, plus a proper forensic method.
Explore the source
- Yourgenome: what is PCR?Wellcome Genome Campus
PCR finds a species without seeing it
6C · Polymerase Chain Reaction (PCR)US Geological Survey scientists develop species-specific PCR assays for environmental DNA in water. An organism can leave cells or fragments behind, so a sample may reveal its DNA…Specification 6.17Read & use the scienceClose article
Why this matters
US Geological Survey scientists develop species-specific PCR assays for environmental DNA in water. An organism can leave cells or fragments behind, so a sample may reveal its DNA even when the animal itself is not seen. Primers select the sequence to amplify, and controls help detect contamination or failed reactions. A positive signal means target DNA was detected under the assay conditions; it does not directly count living animals or prove they are at that exact spot now. This conservation application uses the same amplification principle as the lesson while adding the problem of interpreting tiny traces in a changing environment.
Back to the specification
Pearson Edexcel IAL Biology · Issue 2, February 2021 · Unit 4, pp. 29–32. The application extends beyond required recall.
- 6.17 know how DNA can be amplified using the polymerase chain reaction (PCR)
Use the science
Why can a positive environmental-DNA PCR result not be converted directly into the number of fish in a lake?
Explore a possible response
DNA can persist, move and be shed at different rates; PCR detects a target sequence, not a one-to-one count of living fish.
Explore the source
- Species-specific quantitative PCR assays for eDNAUS Geological Survey