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Gene Technologies

The polymerase chain reaction and gel electrophoresis

  • 3 things to know
  • Components 01, 02 and 03

Overview

Recall questions on the three stages of a cycle and their temperatures, primer design, copy-number calculations and log plots, and how fragment length is estimated from a gel.

What you need to know

Answer each one before you reveal it.

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Key term

Why is the number of copies plotted against cycle number on a logarithmic scale?

Key term

How is the length of an unknown DNA fragment estimated from a gel?

Process

What happens to the DNA during the denaturation stage of PCR?

Questions from this subtopic

Each one with the model answer underneath.

  1. 1

    What is the polymerase chain reaction used for?

    Model answer

    It is used to make a very large number of copies of one specific section of DNA outside the cell, so that a tiny sample becomes enough to analyse.

  2. 2

    State the three stages of one PCR cycle and the temperature used for each.

    Model answer

    One cycle consists of denaturation at about 95 °C, annealing of the primers at about 55 °C and extension at about 72 °C.

  3. 3

    What happens to the DNA during the denaturation stage of PCR?

    Model answer

    The hydrogen bonds between the complementary base pairs break, so the double helix separates into two single strands.

  4. 4

    What is a primer, and why is one needed at each end of the target sequence?

    Model answer

    A primer is a short single strand of DNA complementary to the sequence at one end of the target; it provides the free 3′ end that DNA polymerase must extend from, and one is needed for each strand because the two strands run in opposite directions.

  5. 5

    How do the primers determine which part of the DNA is amplified?

    Model answer

    Only the region lying between the two primer binding sites is copied, so choosing the primer sequences selects the section of the genome that will be amplified.

  6. 6

    Which enzyme is used in PCR, where does it come from, and why does that matter?

    Model answer

    Taq polymerase is used; it comes from Thermus aquaticus, a bacterium of hot springs, and because it is thermostable it is not denatured at 95 °C and so does not have to be replaced after every cycle.

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