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DNA technologies: PCR, electrophoresis and GMOs (SACE Stage 2 Biology)

Syllabus dot point

“Describe the principles of PCR, gel electrophoresis and the production of genetically modified organisms”

9 min read

Quick answer

PCR amplifies a target DNA sequence by repeated cycles of heating and cooling (denaturation, annealing, extension) using primers and a heat-stable DNA polymerase. Gel electrophoresis separates DNA fragments by size using an electric field, with smaller fragments moving further. GMOs are produced by cutting a gene out with restriction enzymes, joining it into a vector with DNA ligase, and inserting it into a host organism.

Jump to a section
  1. What this dot point is asking
  2. PCR (polymerase chain reaction)
  3. Gel electrophoresis
  4. Producing genetically modified organisms
  5. Exam-style questions

What this dot point is asking

You need to describe how each technique works and what it is used for. SACE expects you to link these tools to applications such as DNA profiling, diagnosis and producing useful proteins.

PCR (polymerase chain reaction)

PCR makes millions of copies of a specific DNA sequence in a few hours. Each cycle doubles the amount of target DNA and has three temperature steps:

  1. Denaturation (about 95 degrees Celsius). Heat breaks the hydrogen bonds, separating the double helix into single strands.
  2. Annealing (about 50 to 60 degrees Celsius). Short single-stranded primers bind to the start and end of the target sequence, marking what to copy.
  3. Extension (about 72 degrees Celsius). A heat-stable DNA polymerase (Taq polymerase) adds complementary nucleotides to each template, building new strands.

Repeating the cycle many times amplifies the target exponentially. Taq polymerase is used because it survives the high denaturation temperature.

Why PCR matters

Amplification lets scientists work with tiny starting samples - a single hair, a drop of blood, or trace cells at a crime scene - by producing enough DNA to analyse.

Gel electrophoresis

Gel electrophoresis separates DNA fragments by size.

  1. DNA samples (often cut into fragments by restriction enzymes) are loaded into wells at one end of an agarose gel.
  2. An electric field is applied. Because DNA is negatively charged (due to its phosphate groups), fragments move toward the positive electrode.
  3. The gel acts as a sieve: smaller fragments move faster and travel further, while larger fragments move slowly and stay near the wells.
  4. After staining, the fragments appear as bands, forming a pattern that can be compared between samples.

This produces a banding pattern used in DNA profiling (comparing crime-scene DNA with suspects, or testing relatedness) and in diagnosis.

Producing genetically modified organisms

A genetically modified organism (GMO) has had DNA from another source inserted into its genome. The general method (recombinant DNA technology) is:

  1. Cut. Restriction enzymes cut DNA at specific recognition sequences, releasing the desired gene and cutting open a vector (often a bacterial plasmid). Many restriction enzymes leave sticky ends - short single-stranded overhangs.
  2. Join. The gene and the cut vector have complementary sticky ends, so they pair up; DNA ligase seals them together to form recombinant DNA.
  3. Insert. The recombinant vector is taken up by a host cell (for example, a bacterium).
  4. Express. The host transcribes and translates the inserted gene, producing the desired protein.

A classic application is genetically modified bacteria that produce human insulin for treating diabetes. Other examples include pest-resistant crops and gene therapy research.

Reading a DNA profile gel

Matching a sample to a suspect

A gel shows the crime-scene sample in lane 1 and three suspects in lanes 2 to 4. The crime-scene sample shows bands at fragment sizes that exactly line up with the bands in lane 3, but not lanes 2 or 4.

Because each person's restriction-fragment pattern depends on their unique DNA sequence, a matching banding pattern indicates that suspect 3's DNA is consistent with the crime-scene sample. Smaller fragments sit lower (further from the wells) and larger fragments sit higher, so matching means bands align at the same positions.

Common errors
Saying large fragments travel furthest
Smaller fragments move faster and travel further through the gel.
Forgetting why DNA moves toward the positive electrode
DNA is negatively charged, so it migrates toward the positive electrode (anode).
Confusing the roles of enzymes
Restriction enzymes cut DNA; DNA ligase joins fragments together.
Leaving out primers or Taq in PCR
Primers define the region copied, and a heat-stable polymerase is essential because ordinary polymerase would denature at 95 degrees Celsius.

Exam-style questions

Questions in the style of SACE Board exam questions on this dot point, each with a worked answer. They are written by ExamExplained unless tagged "Past paper"; the year shows the paper a question is modelled on.

SACE 2018-style3 marks
Scientists used CRISPR/Cas9 on colon cancer cells to inactivate (knockout) the GalNAc-T6 gene. Explain why it is necessary to know part of the base sequence of the GalNAc-T6 gene in order to use CRISPR/Cas9 to inactivate the gene.
Show worked answer →

Three linked points earn full marks.

  1. CRISPR/Cas9 is guided to its target by a short guide RNA (gRNA), which must be complementary to part of the target gene's base sequence.

  2. To design a gRNA that base-pairs with the correct site, you must already know that section of the gene's base sequence, otherwise the gRNA cannot be made to match.

  3. Without the correct sequence the gRNA would not bind the GalNAc-T6 gene, so Cas9 would not cut at the right place and the gene would not be inactivated, or an unintended off-target gene could be cut instead.

SACE 2018-style2 marks
CRISPR/Cas9 uses a guide RNA attached to the Cas9 protein. Describe one consequence of the guide RNA being too short.
Show worked answer →

For 2 marks, state the consequence and explain why.

A guide RNA that is too short is less specific because it has fewer bases to pair with the target sequence, so a short sequence is more likely to also be complementary to other sites in the genome.

As a result, Cas9 may bind and cut at the wrong location, an off-target cut, damaging or inactivating a gene other than the intended GalNAc-T6 gene.

SACE 2018-style1 mark
Human proinsulin can be produced by genetically engineered bacterial cells. State one method that could be used to transfer a human gene into bacterial cells.
Show worked answer →

Any one valid recombinant DNA method earns the mark, for example inserting the human gene into a bacterial plasmid (vector) using restriction enzymes and DNA ligase, then having the plasmid taken up by the bacterium (transformation). Naming a plasmid vector, or transformation by heat shock or electroporation, is accepted.

SACE 2019-style1 mark
Identify the technique that could be used to determine the order of the nucleotide bases in a gene, for example to read an electropherogram of a cow gene.
Show worked answer →

DNA sequencing, for example Sanger sequencing or gel electrophoresis of labelled fragments producing an electropherogram. The mark is for naming a technique that reads the base order.

Practise this

Sources & how we know this

Based on the official SACE Board subject outline. Not yet individually reviewed by a person; always check SACE Board and your school's materials for current rules.

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