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6.5.1Unit 6: Practical Skills in Biology II

Polymerase Chain Reaction — IAL Edexcel Linear Revision Notes & Topical Questions

5 min read 6 parts Lesson 17 of 48 Unit quiz

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Part 1 of 6Why PCR Is Used

6.5.1 Polymerase Chain Reaction
DNA profiling
  • Every person except an identical twin has a unique DNA sequence, so a DNA profile can identify individuals.
Small samples
  • Evidence such as saliva, skin, hair or blood may contain very little DNA, which PCR can amplify.
In vitro replication
  • PCR copies DNA outside a living organism, so it is an in vitro method.
Other uses
  • It is also used in genetic engineering and in studying relationships between organisms.

Part 2 of 6Requirements

Requirements
Template DNA
  • The DNA to be amplified; a single molecule is enough.
Primers
  • Short single-stranded DNA sequences complementary to the ends of the target region, which mark where copying begins.
DNA polymerase
  • Taq polymerase from the bacterium Thermus aquaticus builds new strands and does not denature at the high temperature used.
Nucleotides and buffer
  • Free nucleotides supply the building blocks and a buffer keeps the pH at the optimum.

Part 3 of 6The Three Stages of Each Cycle

1
Denaturation: the mixture is heated to about 95 °C, which breaks the hydrogen bonds between the two DNA strands.
2
Annealing: it is cooled to roughly 50–60 °C so that primers bind to the complementary sequences on the separated strands.
3
Elongation: it is heated to about 72 °C, the optimum for Taq polymerase, which adds nucleotides to make a complementary strand.
4
This produces two identical double-stranded molecules from each original molecule, so the amount of DNA doubles.
5
A thermal cycler repeats the stages automatically, usually for 20 or more cycles.

Part 4 of 6Preparing for Electrophoresis

Cutting
  • After PCR the DNA is treated with restriction endonucleases to produce fragments of different lengths.
Labelling
  • Fluorescent tags can be added so that the fragments can be seen under UV light.
Exponential
  • Because each cycle doubles the DNA, n cycles produce 2ⁿ copies from one molecule.
Why Taq
  • Using a heat-stable polymerase avoids having to add fresh enzyme after every heating step.

Part 5 of 6Worked Example

Question

  • A forensic sample contains 50 copies of the target DNA.
  • How many copies are present after 25 PCR cycles, assuming that every cycle doubles the DNA?

  1. The number of copies after n cycles is the starting number × 2ⁿ.
  2. Calculate 2²⁵ = 33 554 432.
  3. Multiply50 × 33 554 432 = 1 677 721 600.

Answer About 1.7 × 10⁹ copies.

Part 6 of 6Exam Checklist & Tips

6.5.1 – Key Points for the Exam

  • PCR Polymerase chain reaction, an in vitro method of amplifying DNA.
  • Primer A short single-stranded DNA sequence that binds to the template and starts copying.
  • Taq polymerase A heat-stable DNA polymerase from Thermus aquaticus.
  • Annealing The binding of primers to complementary sequences on single-stranded DNA.

Common Mistakes

  • Writing that PCR makes protein, when it makes copies of DNA.
  • Mixing the order of the stages; denaturation first, then annealing, then elongation.
  • Giving the elongation temperature as 95 °C, which is the denaturation temperature.
  • Saying that the DNA polymerase is destroyed by the 95 °C stage, which Taq polymerase is not.

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