A2 Biology 9700: PCR Cycle Stages and Gel Electrophoresis

A researcher has only a very small sample of DNA extracted from a plant leaf. Before this DNA can be studied further, the researcher first uses the polymerase chain reaction (PCR) to make many copies of a particular gene, then separates the resulting DNA fragments using gel electrophoresis.

(a) The PCR reaction mixture contains the DNA sample, primers, free nucleotides and a heat-stable DNA polymerase. Describe what happens during each of the three temperature stages of one PCR cycle, stating an appropriate temperature for each stage. [6]

(b) Explain why a heat-stable polymerase, such as Taq polymerase, is used in PCR rather than the DNA polymerase found in human cells. [2]

(c) After 30 cycles of PCR, the amplified DNA is loaded onto an agarose gel and an electric current is applied. Explain how gel electrophoresis separates DNA fragments of different lengths, and explain why the DNA fragments move towards the positive electrode. [4]

Part (a): The three stages of one PCR cycle

Each cycle of the polymerase chain reaction consists of three temperature stages, repeated many times:

Denaturation (about 95 °C). The reaction mixture is heated to a high temperature. This breaks the hydrogen bonds between complementary bases on the two strands of the DNA double helix, separating (denaturing) it into two single strands. This provides a single-stranded template for copying.
Annealing (about 55 °C). The mixture is cooled to a lower temperature so that the short, single-stranded DNA primers can bind, by complementary base pairing, to their specific target sequences flanking the gene of interest, one primer on each single strand. Primers are needed because DNA polymerase can only extend an existing strand; it cannot start a new strand from nothing.
Extension (about 72 °C). The temperature is raised again to the polymerase’s optimum working temperature. Taq polymerase binds at each primer and synthesises a new, complementary strand of DNA by joining free nucleotides in sequence, working along the template strand until it has copied the target gene.

At the end of one cycle, the amount of double-stranded DNA containing the target gene has doubled. Because the cycle is repeated many times, the number of copies of the target gene increases exponentially.

Part (b): Why a heat-stable polymerase is needed

Every PCR cycle begins with a denaturation step at around 95 °C, and this step is repeated many times (often 30 or more cycles) within the same reaction tube. An ordinary DNA polymerase, such as the polymerase found in human cells, would be denatured by this repeated heating: its tertiary structure, including its active site, would be permanently disrupted, so it would stop working after the very first cycle and would need to be replaced before every extension stage.

Taq polymerase is isolated from a thermophilic (heat-tolerant) bacterium and is heat-stable, so it survives repeated heating to 95 °C without losing its shape or function. This means a single addition of Taq polymerase at the start of the reaction is enough for it to keep working throughout all the cycles, making PCR practical to automate.

Part (c): Separating DNA fragments by gel electrophoresis

DNA fragments are loaded into wells at one end of an agarose gel, and an electric current is applied across the gel.

Direction of movement. The sugar-phosphate backbone of DNA carries many phosphate groups, each with a negative charge, so a DNA molecule is negatively charged overall. This means DNA fragments are attracted towards, and move towards, the positive electrode (anode).
Separation by size. As the fragments move through the gel, they must pass through the small pores in the gel matrix. Smaller DNA fragments pass through these pores more easily and so move faster and travel further in a given time than larger fragments, which are slowed down more by the gel matrix.

After the current has run for a set time, the fragments have separated into distinct bands according to their length: the smallest fragments have travelled furthest towards the positive electrode, while the largest fragments remain closest to the wells where the samples were loaded.

Final answers

(a) Denaturation (~95 °C) separates the DNA strands; annealing (~55 °C) allows primers to bind to the target sequence; extension (~72 °C) allows Taq polymerase to synthesise new complementary strands from the primers.
(b) Taq polymerase is heat-stable, so it survives repeated heating to ~95 °C and does not need replacing every cycle, unlike ordinary human DNA polymerase.
(c) DNA is negatively charged (due to phosphate groups) and moves towards the positive electrode; smaller fragments pass through the gel’s pores more easily and so travel further than larger fragments, separating the fragments by size.