Southern blot

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Autoradiogram of a Southern blot

The Southern blot , also known as the Southern blot hybridization method, is a molecular biological test method for DNA developed by Edwin M. Southern in 1975 . It enables the detection of a gene sequence in a complex DNA mixture (e.g. the entire genome of an organism) within a short period of time without having to decipher all the sequences in the mixture.

functionality

The DNA to be examined is treated with one or more restriction enzymes and then separated according to size by gel electrophoresis . The DNA fragments are split into single strands by alkalis and the separation pattern created in the gel is transferred to a membrane (usually nylon or nitrocellulose ) ( blotting ) and permanently fixed there.

The membrane is then treated with a chemically or radioactively labeled gene probe . This probe consists of single-stranded RNA (RNA-DNA hybrids are more stable than DNA-DNA hybrids), which is complementary to the sequence sought, or of double-stranded DNA, which is denatured by heating prior to hybridization. If this sequence is somewhere on the membrane, the probe forms base pairs with it and binds permanently in this area (hybridization process).

All unspecific bindings are then washed off. Depending on the marking of the probe, the detection takes place z. B. by placing an X-ray film and intensifying screens or phospho-imager plates (as autoradiography for radioactive markings) in a light-protected cassette. If the probe is coupled to an enzyme, the enzymatic reaction on the membrane can be detected.

Test procedure

Pre-treatment of the DNA

The DNA to be examined is mostly genomic DNA. It is isolated from the cells and treated with one or more restriction enzymes. Depending on how often the selected enzymes cut in the sequence sought, a different number of detectable fragments are created. If, for example, there is no cleavage point in the sequence, a band is formed; if the enzymes cut once, two bands are formed. (Exceptions here are of course plasmids , in which two bands only arise at two interfaces, since they are initially present as a ring.)

This applies if the sequence you are looking for is only located at one point in the genomic DNA or if, as is almost always the case with a diploid organism, the sequences do not differ in their fragment length. In a quantitative analysis, the signal strength provides information on the number of copies. Therefore, transgenic organisms should be examined by Southern blotting, since the number of signals provides information about the number of integration sites and the internality allows conclusions to be drawn about the number of integrates.

Agarose gel under UV light

Gel electrophoresis

After this pretreatment, between 5 µg and 30 µg of the DNA are separated according to size together with a marker in an agarose gel. Since the restriction enzymes cut randomly in the DNA, there are fragments of every size. In this way, there are no bands in the gel, but an even distribution of the DNA, a smear.

After the electrophoresis , a fluorescent ruler is placed next to the marker on the gel previously stained with ethidium bromide and photographed under UV light . You now know which fragments have run how far in the gel due to their size. This is necessary because the marker is no longer visible after detection with the probe. You can later determine the molecular mass of the fragments that have become visible by simply measuring with a ruler .

The gel is then treated successively with highly diluted hydrochloric acid , a denaturing solution and a neutralizing solution in order to prepare it for the DNA transfer.

Blotting

The transfer of DNA from the gel to a membrane is called blotting . There are different possibilities for this:

Schematic structure of the capillary blotting
Capillary blot
The driving force is a flow of liquid, which starts from a reservoir and runs from below through the gel and on through the membrane to a stack of absorbent material. The liquid is usually an alkaline buffer solution, which breaks the DNA down into single strands. This current pulls the DNA out of the gel, which then gets stuck in the mesh of the membrane. The procedure usually runs for 10 to 12 hours (overnight). During this time, up to 2 liters of saline solution can run through a 15 by 15 centimeter gel. It is important that there are no air bubbles anywhere in the structure, as they interrupt the flow of liquid and the DNA is not transferred at this point.
Vacuum blot
In principle, it works like the capillary blot. Instead of the absorbent material, however, a negative pressure pulls the liquid through the gel and membrane. The vacuum blot is faster and more economical in terms of saline solution.
Electro blot
The electroblot uses the negative charge of the DNA. The gel lies on a cathode plate . The membrane lies on top of the gel and the anode plate above it . A saline solution ensures that an electric current can flow and the DNA moves towards the anode. It migrates out of the gel and gets stuck on the membrane.

After the blotting, the membrane is swirled in a low concentration “salt buffer”. The DNA can then be permanently fixed in the membrane by crosslinking with UV light - crosslinking . If you do not want to continue with the analysis immediately, it is shrink-wrapped and stored in the refrigerator or stored between blotting filter paper at room temperature. Instead of crosslinking, you can “bake” the membrane for 2-3 hours at 80 ° C.

blocking

The remaining after the transfer, free DNA-binding sites on the membrane are usually with a blocking solution with heat-denatured DNA in ten fold concentrated Denhardt solution with the surfactant SDS (1% w / v) in a phosphate - buffer incubated (50 micrograms per milliliter, for five hours at 42 ° C), which saturates the DNA-binding sites on the membrane. For this purpose, a comparatively inexpensive DNA from salmon or herring sperm is usually used. The surfactant reduces unspecific binding of non-DNA molecules to the membrane. The blocking (synonymous with prehybridization ) avoids unspecific binding of the probe over the entire membrane, which would otherwise lead to strong background staining .

Synthesis of the probe

The synthesis of the DNA probe is done either enzymatically by PCR , random priming or nick translation . You present the target sequence and copy it very often, use it as a template or modify it. The modules that will be used later for detection are installed. In the case of an RNA probe, the synthesis takes place by means of in vitro transcription with bacteriophage RNA polymerases , such as the T7 RNA polymerase . The two most common markings are:

α- 32 P dATP mark
The DNA is made up of the four basic building blocks dATP, dTTP, dGTP and dCTP. For this experiment, the dATP carries the radioactive phosphorus isotope 32 P. During the synthesis it is built into the probe, which continuously emits beta rays. Similarly, ATP, GTP, CTP and α- 32 P-UTP are used in RNA synthesis . If you place the membrane on an X-ray film, this is blackened at the points with the probe. A modern alternative is placing it on a phospho-imager screen. This is a special plate, the coating of which is permanently chemically changed by radiation . This change can be read into the computer with a reader and further processed there. The plate can then be regenerated and used again
DIG mark
During copying, dUTP is incorporated into the DNA instead of dTTP at some points. The glycoside dig oxigenin is attached to this building block . DIG-UTP is used analogously for in vitro transcription. You can now bind this digoxigenin with a specific anti-DIG antibody to which an enzyme is coupled. This catalyzes a light or color reaction . The chemiluminescence is detected by placing the membrane on photographic paper. The places where the probe is located are blackened by the resulting light. This method has the advantage that you do not work with radioactive material.

In addition to DIG marking, there are other chemical marking systems, such as the biotin / streptavidin system, in which a visible dye is formed. What all chemical markers have in common is that they are less sensitive than radioactive markers. Nevertheless, they are used in everyday laboratory work when, for example, no isotope laboratory is available.

Hybridization

The probe, usually 50 µl DNA solution, is heated to 94 ° C immediately before use. The heat causes the double-stranded DNA to separate. This is the only way it can base pairing with the DNA on the membrane. The probe solution is then immediately mixed with 5 to 10 ml of a hybridization solution and placed in a glass tube together with the membrane. This tube is automatically turned in an oven at 40 to 60 ° C for several hours. The high temperature guarantees that the probe only binds to the desired target sequence and does not enter into any unspecific interactions with other sequences or membrane parts.

The exact temperature to be selected, however, depends on the length and the G / C content of the probe; also on the salt concentration of the solution. A formula for calculating is as follows: Tm = 81.5 ° C + 0.41 × (% W / C) + 16.6 log [Na +] - 500 / n - 0.61 * (% formamide); where n = number of probe bases; Formamide is a common component of hybridization solutions.

The membrane is then washed several times with salt solutions of low concentration and at temperatures up to 65 ° C in order to completely remove the unbound portion of the probe. The detection follows depending on the marking.

Stripping

Stripping is the process of removing the specifically bound probe. After detection it is possible to immerse the membrane in a stripping solution and heat it to 94 ° C for several minutes. The base pairings between the probe and the target sequence are broken by the heat and the probe becomes detached. The membrane can now be prehybridized again and then treated with another probe. This cycle can be repeated up to 20 times depending on the amount of DNA transferred on the membrane, the quality of the membrane and the solutions.

Similar methods

Analogous to the Southern blot, there is also a Northern blot . Here, RNA is separated by gel electrophoresis and transferred to a membrane. The expression status of a gene can be checked with the Northern blot.

In Western blotting , proteins are separated by a somewhat different electrophoresis (PAGE - polyacrylamide gel electrophoresis ) and transferred to the membrane. Specific antibodies serve as probes. However, one can also investigate protein interactions here, in which case the respective ligand is then detected by antibodies (Far Western blotting). The combination of Western and Southern blots is known as the Southwestern blot .

literature

Further literature

References

  1. ^ EM Southern: Detection of specific sequences among DNA fragments separated by gel electrophoresis. In: Journal of Molecular Biology . Vol. 98, No. 3, 1975, pp. 503-517, PMID 1195397 , doi: 10.1016 / S0022-2836 (75) 80083-0 .
  2. ↑ Judy Meinkoth, Geoffrey choice: Hybridization of nucleic acids Immobilized on solid supports. In: Analytical Biochemistry . Vol. 138, No. 2, May 1984, pp. 267-284, doi: 10.1016 / 0003-2697 (84) 90808-X .

See also

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