In this video, we're going to talk about the Southern blotting technique. Southern blotting is a specific technique used to rapidly detect a specific DNA sequence by using DNA probes. There are other types of blotting that exist. For example, Northern blotting, which is used to detect RNA sequences, and Western blotting, which is used to detect specific proteins. In this video, we'll be mainly focusing on Southern blotting, which is used to detect DNA sequences. Down below, we are showing you the five different steps of Southern blotting that are numbered 1 through 5. These numbers that you see here in the text, 1 through 5, correspond with the numbers that you see down below in the image 1 through 5.

In the first step of Southern blotting, we're going to take some unknown DNA samples and fragment them, and then separate those DNA fragments within the unknown DNA sample by size by using gel electrophoresis, which we covered in previous lesson videos. If you take a look at our image down below at step number 1, you can see here that gel electrophoresis is going to be used to separate fragments of an unknown DNA sample.

In step number 2, we're going to take the DNA on the gel and denature the DNA so that the DNA becomes single-stranded DNA, or ssDNA, for short. This is achieved by incubating the gel with a denaturing buffer. If we look at our image down below, we can get a better understanding of this. The gel from step number 1 is going to be placed into a container that has multiple different components. This container will include the denaturing buffer that will help denature the DNA and create single-stranded DNA. It also contains a sponge on which the gel is placed. Above the gel, a nitrocellulose filter paper, shown in a greenish color, is placed, and on top of this filter paper, paper towels are placed. Essentially, the denaturing buffer will be absorbed upwards through all these components. The buffer denatures the DNA and creates single-stranded DNA because the pH of the buffer will increase, thus causing the DNA to denature.

Now, the filter paper is going to be used to blot the gel, referring to transferring the DNA from the gel to the filter paper. The denaturing buffer will migrate through all these substances to the paper towel stack at the very top. As a result, the DNA from the gel is transferred to the nitrocellulose filter paper and denatured into single-stranded DNA. In step number 3, you can see that the single-stranded DNA is transferred over to the nitrocellulose filter paper as the denaturing buffer is being absorbed and migrating through to the paper towels.

In step number 4, the filter paper is removed and incubated with radioactive probes that are complementary to the specific sequence of interest we want to detect. The nitrocellulose filter paper is placed into a container (shown as a little ziplock bag) and exposed to these DNA probes. The DNA probes, being radioactive and complementary to the sequence of interest, will only bind to specific fragments of DNA.

In the final step, step number 5, the radioactive filter paper can be analyzed, and visible bands on that filter paper are those that bind to the DNA probe in step number 4. If we look at step number 5 down below, you can see that the DNA, complementary to the probe, is visualized. Here, we only have specific sets of bands that are complementary to the probe. Although there are more DNA bands in the filter paper, the only ones that will be visualized are those complementary to the probe. These DNA bands, shown in yellow, are the only DNA bands of all those that were over here that are actually complementary to the probe. Hence, lanes 2 and 4 contain samples that actually have the sequence of interest. This is the fundamental process of how Southern blotting works, and it is used to detect specific DNA sequences complementary to DNA probes. This concludes our brief introduction to Southern blotting, and we will be able to get some practice applying these concepts as we move forward in our course. See you all in our next video.