Southern Blotting - Online Tutor, Practice Problems & Exam Prep

Southern blotting is a technique used to detect specific DNA sequences using radioactive DNA probes. The process involves fragmenting unknown DNA samples, separating them by size through gel electrophoresis, and denaturing the DNA to single-stranded form. The single-stranded DNA is then transferred to a nitrocellulose filter paper, where it is incubated with radioactive probes that bind to complementary sequences. The presence of these sequences is visualized as bands on the filter paper, indicating the samples containing the target DNA sequence.

Southern Blotting

In this video, we're going to begin our lesson on a technique that's called Southern blotting, but before we talk about the actual Southern blotting technique, it's actually first helpful to talk about radioactive probes. And so in this video, we're going to focus on radioactive probes, and in our next lesson video, we'll talk more about the Southern blotting technique. What's important to know is that after cloning a gene, the DNA that was cloned can actually be used as a probe to detect the same sequence in an unknown DNA sample.

A probe or probes are really just radioactively labeled molecules that are visualized using radioactive detection. DNA probes are going to be single-stranded DNA molecules that are going to be complementary to a specific sequence of interest. The DNA probe, which is going to be single-stranded and radioactive, when it is bound to the specific sequence of interest, enables us to detect the presence of a specific sequence of interest by detecting the presence of the radioactive probe.

If we take a look at our image down below, we can get a better understanding of these radioactive probes. In our example, it's saying radioactive probes are used to identify DNA that is complementary to it in different samples. Notice in this image over here on the left-hand side, we have 2 different test tubes with different samples. We have sample A over in the left test tube and sample B over here in the right test tube, and both of these samples contain DNA.

Suppose we want to identify which of these samples has a specific DNA sequence of interest. We can use a radioactive DNA probe, which is going to be a single-stranded DNA molecule as you see here, which is going to be radioactively labeled. We can put this radioactive DNA probe into both of these samples. Under the right conditions, when we incubate the samples with the probe, what will happen is the sample where we can actually detect radioactivity is going to have the sequence of interest, whereas the sample where we do not detect radioactivity will not have the sequence of interest.

In this image, the radioactivity is represented by the yellow color that you see. In terms of having the sequence of interest, because we see radioactivity in sample A, we would say that sample A does have the sequence of interest. But sample B, because we do not detect any radioactivity, would mean that it does not have the sequence of interest. Essentially, by using these radioactive probes, we can detect the presence of a specific sequence.

We'll be able to see how radioactive probes are going to be important in the technique Southern blotting in our next video. But for now, this here concludes our introduction to radioactive probes and we'll be able to get practice applying these concepts as we move forward. So I'll see you all in our next video.

A southern blotting technique is used to detect a specific ______ sequence from a blood or tissue sample.

• A. RNA
• B. DNA
• C. Protein
• D. Lipid

A _____ is a single-stranded DNA molecule used in hybridization reactions to detect the presence of a particular gene in an assortment of DNA fragments.
(Hybridization Reactions:reactions that combine two complementary single-stranded DNA molecules)

• A. Plasmid.
• B. Vector.
• C. Probe.
• D. Blot.
• E. Polymerase.

Steps of Southern Blotting

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.

A geneticist wants to see if her patient has Gene X. The geneticist takes a blood sample from her patient and prepares a southern blot. How will the geneticist know if her patient possesses Gene X?

• A. A radioactive probe, which is complementary to Gene X, will bind to the membrane.
• B. A radioactive probe, which has the same sequence as Gene X, will bind to the membrane.
• C. A radioactive probe will be absent from the membrane.
• D. A southern blot cannot confirm if the patient possesses Gene X.

Place the following steps of Southern Blotting in the correct order.
a) ______:Filter paper is incubated with the labeled DNA probe which anneals to the ssDNA fragments.
b) ______:Analyze gel to determine the presence of the DNA sequence of interest.
c) ______:Separate DNA fragments by size using gel electrophoresis.
d) ______:Denature DNA by soaking gel in a basic solution.
e) ______:Fragment unknown DNA sample(s) using restriction enzymes.

Okay, everyone, place the following steps of Southern blotting in the correct order. Remember, Southern blotting allows us to detect certain sequences of DNA from a DNA sample from an individual. So, what's the correct order of all these steps of Southern blotting? So, what's the first thing that we have to do? Well, we have to take the DNA from the particular person and we have to fragment that DNA, then put that DNA in gel electrophoresis and separate it by size. So the first thing we need to do is e, fragment unknown samples of DNA with restriction enzymes. So this is number 1. Okay, so now I need to take those fragmented DNA samples and put them in gel electrophoresis and separate the DNA fragments by size. So c is number 2. And now that we've separated them by size, what are we going to do? We're going to denature that DNA. We're going to soak it in a basic solution to denature the DNA or take double-stranded DNA and separate it into single-stranded DNA. That's what denature means. So d is number 3. Then we're going to move on to the filter paper step. We're going to take the filter paper and we're going to incubate it with the labeled DNA probe, which anneals to the single-stranded DNA fragments. Remember that the probe is going to complementarily bind to the sequence of DNA that the scientist is interested in. And that probe is labeled radioactively so that the scientists can find that probe. So that's going to be step number 4. And finally, that leaves b as step number 5, which is where the scientists analyze the gel to determine the presence of the DNA sequence of interest, which is either bound to the probe, and they can see it, or the particular person does not have the gene of interest, and the probe doesn't bind to anything. So, this is the correct order of the steps of Southern blotting. Okay, everyone. Let's move on to our next video.

By analyzing the Southern Blot results below, which of the samples contains the gene of interest?

• A. A only.
• B. A & C.
• C. B & D.
• D. A, C, & E.
• E. E only.
• F. None.

Here’s what students ask on this topic:

What is Southern blotting and how does it work?

Southern blotting is a molecular biology technique used to detect specific DNA sequences within a complex mixture. The process involves several steps: First, DNA samples are fragmented and separated by size using gel electrophoresis. Next, the DNA is denatured to single-stranded form and transferred to a nitrocellulose filter paper. This filter paper is then incubated with radioactive DNA probes that are complementary to the target sequence. The probes bind to their complementary sequences, and the presence of these sequences is visualized as bands on the filter paper, indicating which samples contain the target DNA sequence.

What are the steps involved in Southern blotting?

Southern blotting involves five main steps: 1) Fragmenting the DNA samples and separating them by size using gel electrophoresis. 2) Denaturing the DNA on the gel to single-stranded form using a denaturing buffer. 3) Transferring the single-stranded DNA to a nitrocellulose filter paper. 4) Incubating the filter paper with radioactive DNA probes that are complementary to the target sequence. 5) Visualizing the DNA-probe hybrids as bands on the filter paper, which indicate the presence of the target DNA sequence in the samples.

What is the role of radioactive probes in Southern blotting?

In Southern blotting, radioactive probes are single-stranded DNA molecules that are labeled with a radioactive isotope. These probes are complementary to the specific DNA sequence of interest. When the nitrocellulose filter paper containing the single-stranded DNA is incubated with these probes, they bind to their complementary sequences. The presence of the target DNA sequence is then detected by visualizing the radioactive signal, which appears as bands on the filter paper. This allows researchers to identify which samples contain the specific DNA sequence of interest.

How is DNA denatured in the Southern blotting process?

In the Southern blotting process, DNA is denatured to single-stranded form by incubating the gel containing the DNA fragments with a denaturing buffer. This buffer typically has a high pH, which disrupts the hydrogen bonds between the DNA strands, causing them to separate into single strands. The denatured DNA is then transferred to a nitrocellulose filter paper, where it can be hybridized with radioactive probes to detect specific sequences.

What is the purpose of gel electrophoresis in Southern blotting?

Gel electrophoresis is used in Southern blotting to separate DNA fragments based on their size. During this step, DNA samples are loaded into a gel matrix and an electric current is applied. The DNA fragments migrate through the gel, with smaller fragments moving faster and farther than larger ones. This separation allows for the identification and analysis of specific DNA sequences when the fragments are later transferred to a nitrocellulose filter paper and hybridized with radioactive probes.