Southern Blot Concept
Southern blot refers to a Molecular Biology technique through which it is possible to verify whether a specific DNA sequence (gene of interest) is present in the sample. The sample under analysis contains a complex mixture (entire genome of an organism). With this technique it is also possible to obtain information on the molecular mass and the relative amount of that specific DNA sequence.
Its name was given in honor of its inventor, Edwin Southern, an English biologist who developed the technique in 1970 at the University of Edinburgh. The technique relies on the separation of DNA fragments by molecular weight through electrophoresis, followed by transfer to a membrane and detection of the DNA fragment of interest by hybridization with a specific probe.
Procedure:
- DNA extraction from a biological sample (cells or tissue);
- DNA fragmentation, resorting to restriction enzymes;
- Separation of the DNA fragments obtained according to the molecular weight, using agarose gel electrophoresis (on the gel a smear will appear due to the high number of fragments present in each sample);
- Denaturation, or separation, of the DNA double strand; it is necessary to treat the gel where the electrophoresis was carried out with an alkaline solution (usually composed of NaOH – sodium hydroxide). This step is essential since only single stranded DNA has the ability to hybridize to the probe and that some RNA that is still in the sample is destroyed;
- Transfer of the separated DNA in the gel to a nitrocellulose or nylon membrane; it is necessary to place the membrane on the gel and apply evenly distributed pressure on the gel, leading to the binding of the DNA to the membrane. The membrane is then heated (when using a nitrocellulose membrane) or exposed to UV radiation (in the case of the use of a nylon membrane) to permanently attach the DNA to the membrane;
- Membrane treatment with the probe, which is defined as a single stranded DNA or RNA molecule whose sequence is known and complementary to the DNA sequence to be studied, making it possible for hybridization by complementarity between the probe and the sequence of interest. The probe is labeled with radioactive atoms, fluorescent dye or an enzyme that generates signal when it comes in contact with a substrate, allowing the detection of the hybridization sites;
- Membrane washing with the aim of removing all the probe that did not hybridize, remaining on the membrane only the probe that hybridized in totality by base complementarity with the sequence of interest;
- Hybridization pattern detection, where X-ray film autoradiography is used for probes designed with radioactive atoms and fluorescent dye, or a chromogenic detection method in the case of probes designed with enzymes. In the case of no hybridization occurring because the DNA sequence of interest in the sample does not exist, only the molecular marker will emit color.
References:
- Berg J.M., Tymoczko J.L., Stryer L. (2002). Biochemistry (5th edition). W. H. Freeman, New York.
- Cooper G.M. (2000). The Cell: A Molecular Approach (2th edition). Sinauer Associates, Sunderland (MA).
- Griffiths A.J.F., Miller J.H., Suzuki D.T., Lewontin R.C., Gelbart W.M. (2000). An Introduction to Genetic Analysis (7th edition). W. H. Freeman, New York.
- Lodish H., Berk A., Zipursky S.L., Matsudaira P., Baltimore D., Darnell J. (2000). Molecular Cell Biology (4th edition). W. H. Freeman, New York.
