Analyzing DNA by Southern Blot

Detecting Specific DNA Fragments After Separating Them by Size

Jun 15, 2009

Identifying specific pieces of DNA in a complex mixture is readily accomplished using a method developed by Dr. Edwin Southern, and the technique bears his name.

The advent of the recombinant DNA era placed a premium on being able to identify specific fragments of DNA that are contained within a complex mixture. A method for accomplishing this task was reported in 1975 by Dr. Edwin Southern. The method he devised is still used in laboratories today and bears his name, the Southern blot. This technology opened the way for later methods focused on RNA and for protein.

The human genome contains approximately 3 billion base pairs of DNA and roughly 25–30,000 genes. Many other organisms have genomes that are equally as complex. If a researcher wishes to examine only one of these genes, for example to see if its organization has been changed in a particular disease, it is very helpful if the piece of DNA of interest can be examined in a manageably-sized fragment. It is rather difficult to analyze the 23 pairs of chromosomes and all 3 billion base pairs of human DNA at one instant.

Fragmenting DNA

The identification of restriction enzymes made the first task much easier. Restriction enzymes will cut through the DNA double helix only in an area where a specific sequence of base pairs is found. Some restriction enzymes recognize a 4-base sequence, some recognize 6, some recognize 10 or more. The restriction enzyme EcoR1 for instance, recognizes the base sequence GAATTC. If the DNA isolated from cells is digested with a restriction enzyme it will cut the DNA into an assortment of specifically sized pieces. The problem is the gene of interest is probably not the only DNA fragment of that specific size after restriction enzyme digestion. And now it is in the midst of a large mixture of other DNA fragments.

Fractionating DNA Fragments by Size

Using gel electrophoresis the fragments of DNA generated by digestion with a restriction enzyme can be separated according to how big they are. For most experiments, a gel separation matrix can be made out of the compound known as agarose, a complex carbohydrate mixture originally isolated from seaweed. Agarose can be boiled into solution, poured into a special mold, and then allowed to cool to form a gel slab (not unlike making gelatin for dessert). By changing the concentration of agarose in the mixture the gel ends up with smaller or larger pores inside of the solidified matrix (see the article on gel electrophoresis). When the DNA mixture is applied to the gel and placed in an electric field, the fragments will move through the gel mixture at a rate proportional to their size. Small fragments can move fast and go far because they can easily fit through the pores in the slab.

Transferring the DNA Fragments to a Membrane

After the DNA fragments have been separated by size, the gene of interest is probably still in a locale where there are numerous other DNA fragments of the same size. One specific fragment is impossible to identify. This is where Dr. Southern’s method came into play. After treating the gel matrix with a few different solutions, he found that he could use capillary action to drive the DNA out of the gel and onto a membrane filter made of nitrocellulose placed directly against the gel. So, the fragments move perpendicular to their original fractionation. Maintaining their original separation, the fragments migrate out of the gel and then stick to the filter. This can now be handled and used for the ultimate step.

Detection of the Specific DNA Fragment

With the complex mixture of DNA now transferred out of the gel and residing on a membrane filter, the question becomes how to detect the presence of the specific fragment of interest. Using a piece of DNA that is complementary to the DNA fragment of interest, Southern used radioactivity to put a tag into the DNA he was planning to use as “the probe”. By mixing the radioactively labeled probe fragment in a solution with the membrane filter under conditions where complementary pieces of DNA will stick to each other, he was able to localize the radioactive DNA to the spot on the filter where the gene fragment of interest was located. It could now be detected by placing the filter against a sheet of X-Ray film and letting the radioactivity expose an image onto the film.

Before the development of more modern methods for DNA detection such as the polymerase chain reaction, Southern blots were used for everything from disease gene analysis to forensic sciences. Even so, this method is still used widely today.

Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503-17.

The copyright of the article Analyzing DNA by Southern Blot in Scientific Research Methods is owned by Kenneth Rosen. Permission to republish Analyzing DNA by Southern Blot in print or online must be granted by the author in writing.