Nucleic Acid Hybridization: Videos & Practice Problems

Nucleic acid hybridization utilizes the complementary base pairing of nucleic acids, such as adenine (A) with thymine (T) and cytosine (C) with guanine (G). Techniques like Southern blotting detect specific DNA sequences by separating DNA via gel electrophoresis, transferring it to a membrane, and using a labeled DNA probe that binds to the target sequence. Northern blotting serves a similar purpose for RNA. Successful binding is indicated by fluorescence or radioactivity, confirming the presence of the desired nucleic acid sequence.

Nucleic Acid Hybridization

Hi. In this video, I'm going to be talking about nucleic acid hybridization. Hybridization is a process that takes advantage of the fact that nucleic acids, especially complementary nucleic acids, such as A or T, C and G, will form bonds with each other. Knowing that, you can perform two different techniques. The first is Southern blotting, and this is used to detect specific genes in cellular DNA.

How you do this is you take DNA from an organism, and then separate it by gel electrophoresis, which, if you don't remember is fine, but this is going to be separating via mass. Then you transfer that onto some type of paper, and then incubate that paper or set that paper in a solution with a DNA probe. This DNA probe is going to be complementary to a sequence that you're interested in. So, let’s say you need to know if the ATCGGAT sequence is present in the cell. What you do is you take the DNA, you run it, you say, okay, this size will run at 7 base pairs. You take that and then incubate it with a probe that would be TAGCCCTA because it's complementary. And, if it binds, then that means it's there.

You typically fix this probe with some type of fluorescent material, or it can be radioactive material. So if you have that on the paper and you incubate the probe, the probe will bind to the sequence if it's there. And that way, when you detect it you'll see this fluorescence. But if it's not there, that sequence isn't present, it won't bind, and there will be no fluorescence. The same technique can be done with Northern blotting, but this uses RNA instead of DNA.

This is what this looks like: This is an example of Southern blotting. You have two DNA sequences you are interested in looking at. You have one that is 5 kilobases and another that has 23 kilobases. You take, you isolate the DNA, you run it on a gel, which is in this box here, and then you incubate it with a probe that has a complementary sequence. And what you'll see is that if these sequences are present, then you'll see them. Here we have A, the sequence is present at 5, and B, the sequence is present at 23.

That’s Southern blotting and Northern blotting as well. It's just done with RNA for Northern blotting. So with that, let’s now move on.

Which method is used to detect RNA sequences?

• A. Northern blotting
• B. Southern blotting
• C. Western blotting
• D. Eastern blotting

Answer: (A)

Here’s what students ask on this topic:

What is nucleic acid hybridization and how does it work?

Nucleic acid hybridization is a technique that exploits the ability of complementary nucleic acid sequences to form stable double-stranded structures. This process involves the pairing of adenine (A) with thymine (T) and cytosine (C) with guanine (G). In practice, a labeled DNA or RNA probe with a known sequence is used to detect a specific target sequence in a sample. The sample nucleic acids are first separated by gel electrophoresis, transferred to a membrane, and then incubated with the probe. If the target sequence is present, the probe will bind to it, and this binding can be detected through fluorescence or radioactivity, confirming the presence of the desired sequence.

What is the difference between Southern blotting and Northern blotting?

Southern blotting and Northern blotting are both techniques used to detect specific nucleic acid sequences, but they differ in the type of nucleic acid they target. Southern blotting is used to detect specific DNA sequences. It involves separating DNA fragments by gel electrophoresis, transferring them to a membrane, and using a labeled DNA probe to identify the target sequence. Northern blotting, on the other hand, is used to detect specific RNA sequences. The process is similar to Southern blotting but involves RNA instead of DNA. Both techniques rely on the principle of nucleic acid hybridization to identify the presence of specific sequences.

How does gel electrophoresis work in nucleic acid hybridization?

Gel electrophoresis is a technique used to separate nucleic acids based on their size. In nucleic acid hybridization, DNA or RNA samples are loaded into a gel matrix and an electric current is applied. Nucleic acids are negatively charged due to their phosphate backbone and will migrate towards the positive electrode. Smaller fragments move faster and travel further through the gel, while larger fragments move more slowly. This separation allows for the identification of specific sequences when combined with a labeled probe in techniques like Southern or Northern blotting. The separated nucleic acids are then transferred to a membrane for hybridization with the probe.

What are the applications of nucleic acid hybridization?

Nucleic acid hybridization has several important applications in molecular biology and genetics. It is used in Southern blotting to detect specific DNA sequences, which can help identify gene mutations, genetic disorders, and the presence of pathogens. Northern blotting uses hybridization to study gene expression by detecting specific RNA sequences, providing insights into how genes are regulated. Additionally, nucleic acid hybridization is employed in techniques like in situ hybridization to localize specific nucleic acid sequences within tissues or cells, and in microarray technology to analyze gene expression profiles on a large scale.

What is a DNA probe and how is it used in nucleic acid hybridization?

A DNA probe is a short, single-stranded DNA sequence that is complementary to the target sequence of interest. In nucleic acid hybridization, the probe is labeled with a detectable marker, such as a fluorescent dye or radioactive isotope. During the hybridization process, the probe is incubated with the sample nucleic acids that have been separated and transferred to a membrane. If the target sequence is present, the probe will bind to it through complementary base pairing. The binding can then be detected using appropriate methods, such as fluorescence or autoradiography, indicating the presence of the target sequence.