18: Northern blot and Southern blot
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
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
Figure 1 above 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
Thumbnail
"Southern blot membrane.jpg" ↗ by Bojan Žunar is licensed under CC BY-SA 4.0 ↗.
Description: Southern blot membrane.
Author
Katherine Mattaini, Tufts University
- 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. ↵