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How do scientists copy, separate and modify DNA?

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

PCR amplifies DNA through temperature cycles, gel electrophoresis separates fragments by size, and GMOs are made by inserting genes using restriction enzymes, ligase and vectors.

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  1. What this dot point is asking
  2. PCR (polymerase chain reaction)
  3. Gel electrophoresis
  4. Producing genetically modified organisms

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.

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.

Exam-style practice questions

Practice questions written in the style of SACE Board exam questions on this dot point, with worked answer explainers. The year tag is the paper they imitate, not the source.

2018 SACE Stage 23 marksScientists 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.
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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.

2018 SACE Stage 22 marksCRISPR/Cas9 uses a guide RNA attached to the Cas9 protein. Describe one consequence of the guide RNA being too short.
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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.

2018 SACE Stage 21 marksHuman proinsulin can be produced by genetically engineered bacterial cells. State one method that could be used to transfer a human gene into bacterial cells.
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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.

2019 SACE Stage 21 marksIdentify 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.
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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.

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