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Wize University Biology Textbook > Lab Techniques for Biology

Nucleic Acid Detection [Northern and Southern Blots]

Proteomics and Protein/Protein InteractionsPrevious Section
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Southern Blot


Method used to detect specific DNA sequences.
  • Fragments are run through electrophoresis gel to separate them based on SIZE.
  • Fragments are transferred to a membrane.
  • A sequence-specific labeled probe hybridizes the DNA fragment.
  • Radiography or fluorescent imaging used to detect probe.
Photo by CNX OpenStax / CC BY
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Northern Blot

Method used to detect specific RNA sequences.
  • The cell is lysed and the RNA is isolated .
  • The RNA is then run on agarose gel electrophoresis.
Once the agarose gel electrophoresis is complete, all of the RNA has been separated based on size. In order to look at a specific mRNA, a Northern Blot can be performed:
  • A membrane is placed on top of the gel.
  • RNA molecules move from the gel onto the membrane through capillary action.
  • Because we know the sequence we are looking for, we can design a probe that is complementary and antiparallel to the sequence. of mRNA we are interested in. This probe is also radioactively labelled.
  • The membrane is incubated with the probe, and the probe will hybridize to the sequence of interest.
  • The membrane is washed and then radioactivity is measured.
Photo by Ilewieszoośmiornicach / CC BY

Practice: Hybridization

The ability of nucleic acids to hybridize to each other is important for which of the following methods? (Select all the apply)
Extra Practice
Practice: Gene Expression
Which technique would allow you to determine whether the transcription from the CFTR gene was compromised in samples obtained from patients with cyctic fibrosis?
Which technique would allow you to determine whether the transcription from the CFTR gene, was compromised in samples obtained from patients with cyctic fibrosis?
Interpreting and drawing Northern blots
Using the following Northern blot, what can we conclude about the gene expression of Hsp1 in high temperatures? Fill in what you predict to be the banding pattern of Hsp1 and 5S RNA levels at 20oC.
You want to measure the rate of Hsp1 mRNA decay at 55oC. Design an experiment to answer that question using a Northern blot as your read-out.
Practice: Gene Expression
Which technique would allow you to determine whether the transcription from the CFTR gene was compromised in samples obtained from patients with cyctic fibrosis?
Which of the following can be resolved using Western blotting?
1. Protein-protein interaction
2. Expression of a protein when induced with a drug
Use the following SDS-PAGE and Western Blot results for this 2 part question:
You are running an experiment where you want to determine if in a eukaryotic cell the rate of an mRNA transcript being degraded. Which of the following describes the experiment and technique you will use?
Unicellular organisms have the ability to change their membrane fluidity depending on temperature. A bacteria is able to survive at -10oC. At this temperature, what would you expect to see on a Northern blot probing for desaturase?
A heat shock protein increases in abundance after exposure to 40°C compared to 24°C. You can visually see the increase in band intensity on a Western blot. You treat your cells with MG132 to block the activity of the proteasome as well as treating the cells with antinomycin to block transcription. Looking at your Western blot, the protein abundance of the heat shock protein decreases over time at the same temperature. What is the most likely reason for this observed decrease?
Interpreting and drawing Western blots
Using the following Western blot, what can we conclude about the protein abundance of HSP1?
What would you add to the preceding experiment to measure the rate of HSP1 degradation?
What would you add to the preceding experiment to measure the rate of HSP1 translation?
Interpreting and drawing Northern blots
Using the following Northern blot, what can we conclude about the gene expression of Hsp1 in high temperatures? Fill in what you predict to be the banding pattern of Hsp1 and 5S RNA levels at 20oC.
You want to measure the rate of Hsp1 mRNA decay at 55oC. Design an experiment to answer that question using a Northern blot as your read-out.
You are studying an olfactory receptor in two strains the roundworm C. elegans. In the wildtype strain, the worms are able to smell attractive food odors. In the mutant strain, the worms are unable to sense when food is nearby. You believe that a severe DNA mutation has led to a deletion of the olfactory receptor gene from the mutant C. elegans worm's genome. How would you detect whether this specific DNA sequence is present in the mutant C. elegans genome?
Which technique would allow you to determine whether the transcription from the CFTR gene, was compromised in samples obtained from patients with cyctic fibrosis?
Which technique would allow you to determine whether the transcription from the CFTR gene, was compromised in samples obtained from patients with cyctic fibrosis?
You are studying an olfactory receptor in two strains the roundworm C. elegans. In the wildtype strain, the worms are able to smell attractive food odors. In the mutant strain, the worms are unable to sense when food is nearby. You believe that a severe DNA mutation has led to a deletion of the olfactory receptor gene from the mutant C. elegans worm's genome. How would you detect whether this specific DNA sequence is present in the mutant C. elegans genome?
In Northern blotting, electrophoresis is used to resolve which biological molecules? What type of probe is used to identify the target molecule(s)?
Source of this question: Griffiths AJ, et al. An introduction to genetic analysis. 11th Ed.