Structural Biochemistry/DNA recombinant techniques/Southern Blot



Southern blotting, which is named after its inventor, Edwin Southern, is a common technique used in molecular biology to separate and characterize DNA. It is an effective way to identify a specific DNA pattern by the following procedures. Southern blotting is a technique used to determine the presence of a specific DNA sequence within a mixture via agarose gel electrophoresis.

Southern blotting is a hybridization technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA.

Southern Blotting was the first technique of its kind. Soon after however, additional analogous techniques known as Western Blotting and Northern Blotting (among others), were cleverly named eponymously; to follow closely follow the convention behind the naming of Southern Blotting. These techniques don’t detect the presence of DNA, but rather the presence of Proteins and RNA, respectively.

Protocols
DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through an agarose or acrylamide gel. The DNA is denatured and transferred from the gel to a solid support. The reason for transferring the DNA fragments to a solid support (usually a nitrocellulose plate) is that the DNA is inaccessible to DNA probes while embedded in the gel. The relative positions of the DNA fragments are preserved during their transfer to the filter. The DNA fragments attached to the filter are then exposed to a strand of radioactively-labeled DNA that is complimentary to the DNA strand on the plate that is of interest. Autoradiography is then used to locate the positions of bands complementary to the probe.

Method


1. ''The DNA sample, if necessary, is digested with an appropriate restriction enzymes, or restriction endonucleases. The digest is then separated by gel electrophoresis, usually on an agarose gel''. If a large amount of restriction fragments are present in the sample, the sample may likely appear as faint smears rather than discrete bands. By the 1970 the DNA fragments was isolated by using gravity. After that time, the scientists found other way to separate DNA fragment. That is Gel Electrophoresis. Electrophoresis uses electricity to separate different sized molecules.
 * Gel Electrophoresis:

2. The digest is then denatured to allow for transfer onto a membrane. Since the sample DNA is double-stranded and only single-stranded DNA can be transferred, the sample must be denatured by soaking in an appropriate alkaline solution (e.g. 0.5M NaOH). If the sample is still too large to be transferred (more than 15kb), the sample may be treated with an appropriate acid to depurinate (e.g. remove the purines) the sample and break it down into smaller fragments. The sample is then neutralized before continuing with the procedure.

3. The sample is transferred onto a membrane which is a sheet of special blotting paper for analysis. The transfer to another membrane is performed in order to preserve the position of the DNA fragments once electrophoresis has been performed. A Nitrocellulose membrane is generally used, though some may prefer the use of nylon for a better binding capacity. It may also be noted that nylon is less fragile than nitrocellulose. The membrane used is laid on top of the gel, and usually paper towels are placed on top of the membrane to ensure an even distribution by applying pressure evenly. The transfer is done usually be capillary action, which may take several hours. Alternatively, a vacuum apparatus can be used; this is very similar to capillary action, though transferring via vacuum apparatus may be faster. The transfer can also occur by moving the DNA out of the gel and onto the membrane by electrophoresis, a process called electrotransfer.

4. The sample is then treated with UV light to irreversibly cross-link the sample to the membrane covalently. Alternatively, the sample may be baked at around 80°C for several hours (this should only be done if using nylon membrane since nitrocellulose is highly combustible).

5. ''The membrane is then probed with labeled, single-stranded DNA (this is the target DNA sequence). This process is also known as hybridization.'' The labeled DNA binds to the membrane DNA via the binding of complementary strands. The label is generally a 32P probe label, though a bioluminescent probe or biotin/streptavidin may also be used. The reason that hybridization is important is that it allows you to physically see it. It should be noted that prior to hybridization, a prehybridization process should be run to restrict the labeled DNA binding to specific sites, because otherwise the labeled DNA may bind to unwanted or unimportant sites within the sample. To do so, salmon sperm DNA treated with varying concentrations of SSC, SDS, and formamide is used. Treat the dsDNA fragment that you are using as a probe with a limiting amount of Dnase, which causes double-stranded nicks in DNA. Add 32P, dATP, and other dNTPs to DNA polymerase I, which has 5' to 3' polymerase activity and 5' to 3' exonuclease activity.
 * 32P labeled:

6. The results are analyzed. If a 32P labeled probe was used, the sample will be analyzed with an autoradiograph, or the use of an X-ray film to reveal whether or not the sample contained the target DNA sequence. If the target sequence is indeed present, the X-ray film will show blackened bands caused by beta emissions from the 32P radiolabeled probe. Sequences in the sample that are very similar to the target sequence will also be shown. If a bio-luminescent probe was used, the luminescence will be a method of visualization. If biotin/streptavidin was used, the sample is analyzed by colorimetric methods, or by looking at the development of color on the membrane.

Method Summary : First, a mixture of restriction fragments is separated by electrophoresis through an agarose gel, denatured to form a single stranded DNA, and then transferred to a nitrocellulose sheet. The positions of the DNA fragments in the gel are preserved on the nitrocellulose sheet because it is directly blotted onto the sheet from the agarose gel. Next, the fragments are exposed to a 32P-labeled single stranded DNA probe which hybridizes with a restriction fragment having a complementary sequence. After hybridization, an autoradiography reveals the position of the restriction-fragment-probe duplex and ultimately the identity of the DNA fragment.

Examples of Southern Blots
Southern Blot done by Nisha Patel, UCSD Pediatrics, Sander Lab Photo uploaded by: Patrick Phuong, UCSD, Sander Lab

As mentioned above, Southern blots utilize DNA probes to detect the presence of complementary, target DNA sequences. In this case, the Southern blot was run using mouse DNA from mice which were breed in the mouse colony. Southern blotting is a way to track the genetic make up of each new litter of mice and the genotypes of old mice. In the picture, it can be seen that heterozygous, mutant, and wild-type mice are all distinguished by the types of bands that show up in their segment of the blot. Heterozygous mice have sequences for both the top and bottom bands whereas mutants only have the bottom band. Wild-type mice have only the top band of DNA detected by the probe. On the left side is a DNA standard, which is run to show the progress of different sizes of DNA. The labeling at the top shows the mouse line and female number for the mouse that gave birth to the litter whose DNA is being blotted.

Uses of Southern blot
By using this technique, we are able to detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavage of the entire human genome with a restriction enzyme. In such a complex mixture, many fragments will have the same or nearly the same length and thus migrate together during electrophoresis.

The ability to determine whether or not a specific sequence of DNA is present in a DNA sample can be applied for several uses. For example, Southern blot can be used to determine the presence and amount of a certain gene within the genome of a certain organism, which also reveals the molecular weight of the specific fragment. Due to the procedures involved in the method, the presence of restriction sites for particular restriction endonucleases can also be determined. It can also be used to compare the degree of similarity between the probe sequence and the sample.

Southern Blotting can also be used to follow the inheritance of selected genes. Mutations within restriction sites change the sizes of restriction fragments and as a result, the positions of bands in the Southern-blotting analysis and autoradiography also change. This change in position can later be compared to normal blot-analyses in order to reveal where the possible change has occurred. The existence of genetic diversity created by these mutations in a population, is termed polymorphism. The detected mutation in turn may have different effects. It may cause disease or it may be closely linked to one that does. Some examples of such diseases include sickle-cell anemia, cystic fibrosis, and Huntington chorea. All of these mutations can be detected by comparing the restriction-fragment-length polymorphisms with normal fragments of DNA.

Common Misunderstandings/Differences
As previously mentioned, Southern Blotting gave rise to other analytical methods such as Western and Northern Blotting, corresponding to Protein and RNA detection, respectively. Often time, the names of the techniques are erroneously interchanged, or even more importantly, are assumed to use the same method components [e.g. instrumentation, chemicals, etc.]. It is important to recognize that although the methods are very similar, they differ in that each are technique for a specific macromolecule; and therefore are treated differently.

To give a brief example, consider the basic differences of a Southern Blot to a Northern Blot. While both use autoradiography in the final steps to visualize their fluorescent labels, their labels are actually different. A southern blot uses a 32P-label DNA probe to interact with a specific sequence of DNA, whereas Western Blot uses radiolabeled antibodies that will interact with the specific protein. Other examples include different types of sheets to which the macromolecules are transferred to after electrophoresis [Nitrocellulose For Southern; Polymer For Western], to even the type of gel used for the electrophoresis step itself [Agarose For Southern; SDS For Western]. To avoid confusion, Southern, Northern, and Western Blots can also be known respectively as: DNA, RNA, or Protein Blots.