17: Northern blot and Southern blot (2024)

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    Summary

    Northern blot (RNA) and Southern blot (DNA) are methods used to detect the presence of a particular nucleic acid sequence in a mixture.

    Also known as

    No alternative names

    Caution

    Nomenclature note: The Southern blot is named after its inventor, Edward Southern, and therefore, the word “Southern” is a proper noun and should always be capitalized in its name. However, the northern blot and western blot were named in reference to the Southern blot and the cardinal directions. Therefore, the words “northern” and “western” in the method names are not proper nouns, and should not be capitalized.

    Samples needed

    Mixtures of nucleic acids

    Method

    The overall methods for northern and Southern blots are very similar, although they are used to detect different molecules, RNA and DNA, respectively. The first step is to separate the nucleic acid mixture by gel electrophoresis. Following electrophoresis, the nucleic acids are transferred to a membrane, then the membrane is treated with a single-stranded nucleic acid probe. If using double-stranded nucleic acid samples, a denaturing step is required to expose the nitrogenous bases.

    The probe has two key features. First, it is able to base pair with the DNA or RNA sequence of interest. Second, it is labeled for visualization, often either by a radioactive or fluorescent molecule. When the membrane is visualized, the signal indicates the size (due to the electrophoresis step), and to some extent, the amount of the nucleic acid sequence of interest.

    Controls

    Ideally, there should be positive and negative controls to test the specificity of probe binding. The electrophoresis step should also include molecular weight ladders.

    Interpretation

    17: Northern blot and Southern blot (2)

    Figure 1above shows the results of a two Southern blots, the first from embryonic stem cells that the authors were attempting to genetically modify, and the second from the resulting genetically modified mice. Importantly, the ES cells being used already had a modification that allowed researchers to easily insert a gene of interest. Therefore, when the genomic DNA was cut with the restriction enzyme SpeI, three band sizes were possible. The wild type locus is shown by the 6.2 kb band, the initial modification (to allow a transgene to be inserted) is shown by a 6.7 kb band, and the locus with the gene of interest (PHGDH) successfully inserted is shown by a 4.3 kb band.

    In panel B, ES cell clones D2, D6, and D7 show one wild type locus and one modified locus without PHGDH (bands at 6.2 and 6.7 kb). Clones D4 and D5 show one wild type locus and one locus with PHGDH (bands at 6.2 and 4.3 kb). Clone D1 has a band at an unexpected size, so that would not be a good clone to select to use to make genetically modified mice. Panel C shows that, as expected, mice that have been PCR genotyped as having two copies of PHGDH (“tetO/tetO”) have only the 4.2 kb band, mice with no transgene (“+/+”) have only the 6.2 kb band, and mice with one copy of PHGDH (“tetO/+”) have both bands.

    Note that when all of the DNA in a mouse cell is digested with SpeI, a huge smear of DNA would be seen on a DNA gel if a normal gel visualization method like ethidium bromide were used. The use of a probe complementary to the locus of interest allows the researchers to see only the bands they care about, not every single SpeI fragment.

    Image Descriptions

    Figure 1image description:

    Two Southern blots.

    Results of first Southern blot
    D1 D2 D4 D5 D6 D7
    Band >6.7 kb x
    6.7 kb x x x
    6.2 kb x x x x x x
    4.3 kb x x x
    Results of second Southern blot
    tetO/tetO tetO/+ +/+
    6.2 kb x x
    4.3 kb x x

    Thumbnail

    "Southern blot membrane.jpg"↗byBojan Žunaris licensed underCC BY-SA 4.0↗.

    Description:Southern blot membrane.

    Author

    Katherine Mattaini, Tufts University

    1. Mattaini, K. R., M. R. Sullivan, A. N. Lau, B. P. Fiske, R. T. Bronson, and M. G. Vander Heiden. 2019. Increased PHGDH expression promotes aberrant melanin accumulation. BMC Cancer 19:723.
    17: Northern blot and Southern blot (2024)

    FAQs

    What is Northern blot and Southern blot? ›

    Northern blot is used to detect a specific RNA sequence in a sample of mixed RNAs. Southern blot is used for detecting a specific DNA sequence in a sample of mixed DNA. Western blot is used to identify a specific amino acid sequence in a sample of mixed proteins. A specific RNA sequence is detected.

    What is the difference between Northern blotting and Southern blotting quizlet? ›

    Southern blot is used to detect DNA. Northern blot is used to detect RNA.

    What does a Southern blot tell you? ›

    Southern blot analysis can be used to investigate whether a gene is amplified, deleted, or structurally rearranged in cancer cells as compared to normal cells. Although this technique is quite labor-intensive, it's particularly useful for detecting large deletions in tumor genomes.

    What is a Southern blot for RNA? ›

    Fragments of DNA and RNA molecules separated by gel electrophoresis are transferred to a nylon or nitrocellulose membrane in a process termed as Southern and Northern blotting, respectively. Southern blotting was introduced by Edwin Southern in 1975 as a method to detect specific sequences of DNA in DNA samples.

    What is a Southern blot for gene expression? ›

    Southern blotting can be applied in studying structure of a gene or to elucidate restriction enzyme maps. Particularly, Southern blotting can be used in the identification of the methylated sites present in case of some genes in particular.

    What is Northern blotting for dummies? ›

    Northern Blotting is a technique used for the study of gene expression. It is done by detection of particular RNA (or isolated mRNA). mRNA is generally represented as 5% of the overall RNA sequence. This method reveals the identity, number, activity, and size of the particular gene.

    What are the advantages of Northern blotting? ›

    The advantages of using Northern blotting include the use of probes with partial homology, the quality, and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and re-probed for years after blotting [41].

    What is the difference between a dot blot and a Southern blot? ›

    For dot blot hybridization, DNA or RNA is spotted directly onto a membrane, while for Southern or northern blot hybridization DNA fragments or mRNAs, respectively, are transferred to the membrane after size separation on an agarose gel by capillary-, vacuum-, pressure-or electroblotting and subsequently hybridized with ...

    What do Northern Blot results tell you? ›

    Northern blot analysis can be used to investigate whether a gene is overexpressed or underexpressed in cancer cells as compared to normal cells. Overexpression means the cancer cells produce or transcribe too much RNA from the gene. Overexpression can occur when a gene is amplified or turned on or turned up.

    Why is it called Northern blotting? ›

    The Northern blot method was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University (Alwine, et al, 1977). Northern blotting takes its name from its similarity to the first blotting methodology, the Southern blot, which is named after the scientist Edwin Southern (Alberts et al., 2008).

    What is the principle of Southern blot? ›

    Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel.

    What is the difference between northern blot and Southern blot? ›

    Southern blot detects specific DNA sequences, Northern blot detects particular RNA sequences, and Western blot detects specific proteins. All these techniques are complementary.

    What is the purpose of blotting? ›

    https://app.monstercampaigns.com/c/yrirrqigyss1ydr...Blotting techniques are used to detect and analyze three types of biological macromolecules: DNA, RNA and proteins. Results of a blotting experiment tell you whether a macromolecule of a specific sequence is present in your sample or not.

    Why is PCR better than Southern blotting? ›

    Southern blotting was invented before PCR, but PCR has replaced blotting in many applications because of its simplicity, speed, and convenience. Following the development of the Southern blot, other types of blotting techniques were invented.

    What is Northern blot for dummies? ›

    Northern blot is a laboratory analysis method used to study RNA. Specifically, purified RNA fragments from a biological sample (such as blood or tissue) are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments.

    What is the primary purpose of northern blotting? ›

    Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment.

    What is the difference between a dot blot and a Northern blot? ›

    For dot blot hybridization, DNA or RNA is spotted directly onto a membrane, while for Southern or northern blot hybridization DNA fragments or mRNAs, respectively, are transferred to the membrane after size separation on an agarose gel by capillary-, vacuum-, pressure-or electroblotting and subsequently hybridized with ...

    Why is it called a Northern blot? ›

    The Northern blot method was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University (Alwine, et al, 1977). Northern blotting takes its name from its similarity to the first blotting methodology, the Southern blot, which is named after the scientist Edwin Southern (Alberts et al., 2008).

    References

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