Southern blot | Biomolecules | MCAT | Khan Academy

Summary

This video script explains the Southern Blot technique, which is used to detect a specific DNA sequence. The process involves cleaving DNA into smaller fragments, separating them by size and charge using gel electrophoresis, transferring the DNA to a filter, and then exposing it to a radio-labeled complementary DNA sequence. The final step is to expose the filter to x-ray film to visualize the radio-labeled DNA, confirming the presence of the gene of interest.

Takeaways

• 🧬 **Southern Blot Overview**: The Southern Blot is a technique used to visualize a specific piece of DNA within a larger sample.
• 🔍 **Identifying Gene A**: The process aims to identify whether a particular gene, Gene A, is present in a given DNA sample.
• ✂️ **DNA Cleavage**: The first step involves cleaving the DNA into smaller fragments using enzymes.
• 🧪 **Gel Electrophoresis**: DNA fragments are then separated by size and charge through gel electrophoresis.
• 🚀 **Fragment Separation**: The gel electrophoresis results in the DNA fragments being arranged by size on the gel.
• 📄 **Transfer to Filter**: The separated DNA on the gel is transferred onto a more robust filter for further processing.
• 🔒 **DNA Hybridization**: A radio-labeled complementary DNA sequence to Gene A is used to hybridize with the DNA on the filter.
• 📡 **Radio-Labeled DNA**: The radio-labeled DNA will bind specifically to Gene A if it is present on the filter.
• 🎞️ **Visualization with X-Ray**: The final step involves exposing the filter to X-ray film to visualize the radio-labeled DNA that has bound to Gene A.
• 🔎 **Presence Confirmation**: The appearance of the radio-labeled DNA on the X-ray film confirms the presence of Gene A in the original DNA sample.

Q & A

What is a Southern Blot?

A Southern Blot is a technique used to visualize a specific piece of DNA that the researcher is interested in, by transferring DNA fragments separated by gel electrophoresis onto a filter and then using a radio-labeled probe to detect the presence of a particular gene.

Why is it necessary to cleave the DNA in the Southern Blot process?

Cleaving the DNA is necessary to break it down into smaller fragments. This allows for the separation of these fragments based on size and charge during gel electrophoresis, which is a crucial step in identifying the presence of a specific gene.

What role do enzymes play in the Southern Blot procedure?

Enzymes are used to cleave the DNA into smaller pieces. They act on the DNA strands, cutting them at specific sequences, which results in a multitude of smaller DNA fragments that can be further processed in the Southern Blot.

How does gel electrophoresis help in the Southern Blot process?

Gel electrophoresis is used to separate the DNA fragments based on their size and charge. The smaller fragments move faster through the gel, allowing for a clear separation that is necessary for the subsequent steps of the Southern Blot.

What is the purpose of transferring the DNA fragments onto a filter in a Southern Blot?

Transferring the DNA fragments onto a filter serves to stabilize the DNA for further analysis. The filter is more robust than the gel, making it easier to handle and allowing for the detection of specific DNA sequences using a radio-labeled probe.

Why is a radio-labeled DNA probe used in the Southern Blot?

A radio-labeled DNA probe is used because it can bind specifically to its complementary DNA sequence. By using a probe that is complementary to the gene of interest, researchers can detect the presence of that gene in the DNA sample.

How does the radio-labeled DNA probe anneal to the gene of interest on the filter?

The radio-labeled DNA probe anneals to the gene of interest by forming hydrogen bonds with the complementary DNA sequence on the filter. This specific binding allows for the detection of the gene of interest.

What is the final step in visualizing the radio-labeled DNA in a Southern Blot?

The final step in visualizing the radio-labeled DNA is to expose the filter to an x-ray film. The radioactivity of the labeled DNA causes it to appear on the film, providing a visual confirmation of the presence of the gene of interest.

What does the appearance of the radio-labeled DNA on the x-ray film indicate?

The appearance of the radio-labeled DNA on the x-ray film indicates that the gene of interest is present in the DNA sample. The specific band or spot on the film corresponds to the location of the gene on the filter.

How does the Southern Blot technique differ from other blotting techniques like Northern and Western blots?

While Southern Blot is used for DNA, Northern blotting is used for RNA and Western blotting is used for proteins. Each technique involves a similar principle of transferring molecules onto a membrane for detection, but the specifics of the process and the types of molecules analyzed differ.

Outlines

00:00

🧬 DNA Visualization with Southern Blot

This paragraph introduces the Southern Blot technique, which is used to visualize a specific piece of DNA, such as Gene A, within a mixture. The process involves several steps: First, the DNA is cleaved into smaller fragments using enzymes. Then, these fragments are separated by size and charge through gel electrophoresis. Following this, the DNA fragments are transferred onto a filter to stabilize them. The next step involves exposing the filter to a radio-labeled DNA probe that is complementary to the gene of interest. Finally, the filter is exposed to x-ray film to visualize the radio-labeled probe, indicating the presence of the gene within the DNA mixture.

05:00

🔍 Confirming Gene Presence with Radio-Labeled Probe

The second paragraph explains the final steps of the Southern Blot process, focusing on the use of a radio-labeled DNA probe to confirm the presence of a specific gene, Gene A, in the DNA mixture. If the radio-labeled DNA binds to the DNA fragment on the filter, it indicates that Gene A is present. The binding is visualized by exposing the filter to x-ray film, which will reveal the location of the radio-labeled DNA, thus confirming the presence of the gene of interest.

Keywords

💡Southern Blot

Southern Blot is a technique used in molecular biology to detect specific DNA sequences. It involves transferring DNA from an agarose gel to a membrane, which is then probed with a complementary, labeled DNA sequence. In the video, Southern Blot is the central method being explained, with the aim of visualizing a specific piece of DNA, such as Gene A, within a mixture.

💡DNA

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. It is composed of a long chain of nucleotides, each containing a sugar molecule, a phosphate group, and a nitrogenous base. In the video, DNA is visualized as a cup filled with a mixture of different DNA sequences, from which the presence of a specific gene is to be determined.

💡Cleave

To cleave DNA means to cut it, typically using enzymes known as restriction enzymes that recognize specific sequences and cut the DNA strand at those points. In the context of the video, cleaving is the first step in the Southern Blot process, where the DNA is cut into smaller fragments to facilitate analysis.

💡Gel Electrophoresis

Gel electrophoresis is a method used to separate mixtures of DNA, RNA, or proteins by size and charge. It involves applying an electric field across a gel matrix, causing the molecules to migrate at different rates based on their size and shape. In the video, gel electrophoresis is used to separate the DNA fragments based on size after the DNA has been cleaved.

💡DNA Fragments

DNA fragments are the smaller pieces of DNA that result from the cleaving process. They are the subject of analysis in techniques like Southern Blot. The video script describes how these fragments are created and then separated using gel electrophoresis before being transferred to a filter.

💡Filter

In the context of Southern Blotting, a filter is a porous membrane that is used to transfer DNA from the gel to a solid support for further analysis. The video explains that after the DNA fragments are separated by size on the gel, they are transferred onto a filter, which is more stable and easier to handle than the gel itself.

💡Radio-labeled DNA

Radio-labeled DNA refers to DNA molecules that have been tagged with a radioactive isotope, allowing them to be detected by their radioactivity. In the video, the radio-labeled DNA is the complementary sequence to the gene of interest (Gene A) and is used to probe the filter to see if the gene is present in the DNA sample.

💡Anneal

Annealing in molecular biology refers to the process where two complementary strands of DNA or RNA come together and form a double-stranded molecule through hydrogen bonding. In the video, the radio-labeled DNA anneals to the DNA fragment that contains the gene of interest, which is a crucial step for the detection of the specific gene.

💡X-ray Film

X-ray film is a type of photographic film that is sensitive to X-rays and can be used to capture images. In the context of the video, the filter with the annealed DNA is exposed to X-ray film to visualize the radio-labeled DNA that has bound to the gene of interest, thus indicating the presence of that gene in the original DNA sample.

💡Gene A

Gene A is the specific gene that the video's narrator is interested in detecting within the DNA sample. It serves as an example of a target gene in a Southern Blot analysis. The entire process described in the video is aimed at determining whether Gene A is present within the mixture of DNA fragments.

Highlights

Introduction to Southern Blot technique for visualizing specific DNA sequences.

Visualizing a specific gene within a mixture of DNA using Southern Blot.

Step 1: Cleaving DNA into smaller fragments using enzymes.

Step 2: Separating DNA fragments by size and charge through gel electrophoresis.

The process of gel electrophoresis and its role in DNA fragment separation.

Step 3: Transferring DNA from the gel to a filter for stabilization.

The importance of using a filter for visualizing DNA fragments.

Step 4: Exposing the filter to radio-labeled DNA complementary to the gene of interest.

The concept of radio-labeled DNA and its use in identifying specific genes.

Step 5: Visualizing the radio-labeled DNA using x-ray film.

The final step of Southern Blot process and its outcome.

How the presence of a specific DNA fragment on x-ray film indicates the presence of the gene of interest.

The significance of Southern Blot in genetic research and diagnostics.

Practical applications of Southern Blot in identifying specific genes within DNA samples.

The innovative aspect of using radio-labeled DNA for gene detection.

The potential impact of Southern Blot on genetic disease diagnosis and treatment.

The theoretical contribution of Southern Blot to molecular biology.

Transcripts

- So in this video, I'm gonna be talking about

something known as a Southern Blot.

So, a Southern Blot basically allows you

to visualize a specific piece of DNA

that you're interested in.

So let's imagine that we have a cup

and it's filled with DNA.

So it's got just a whole bunch of DNA inside.

And there's just lots and lots of those DNA

and let's imagine that I'm specifically

interested in one gene.

So let's imagine that I'm interested in Gene A

and I want to see if Gene A

is inside of this cup.

If it's inside of this long piece of DNA.

Now, in order to figure out whether

or not Gene A is inside this cup,

basically we have to do this process

known as a Southern Blot.

And we'll break it up into a couple of different steps.

So Step 1, what we're gonna do

is we're gonna take this DNA

and we're gonna cleave this.

So, "take the DNA and cleave it."

So, let me draw that out.

So, we're gonna take this big old strand.

We're gonna remove it outside of the cup over here.

So, we got this big strand

and we're gonna cut it up.

We're gonna expose it to enzymes

that will basically cleave the DNA

in a whole bunch of different parts.

And that will result in lots

of these smaller pieces of DNA.

So that's basically the first step.

So we got a bunch of small little pieces of DNA.

Now Step 2, what do we do?

Well, what we're gonna do is we're gonna

take all these tiny little DNA fragments

and we're gonna run them on the gel.

So, specifically we're gonna do

a gel electrophoresis, "electrophoresis"

on these DNA fragments.

And I made a video on gel electrophoresis

if you want to refresh,

you can watch that video.

But basically, the gel electrophoresis

will help us separate these DNA fragments

based on size and based on charge.

So, let's just diagram that out.

So, we're gonna take these DNA fragments

and we're gonna run them on a gel.

So, let's imagine that this is the gel

and we add the DNA fragments to different wells.

So the fragments are gonna move down the gel

and they're gonna basically be separated

based on size and based on charge.

So, we're gonna have these fragments separated like so.

So now, we've got this gel

and we've got the DNA fragments

separated by size on this gel.

So the next step, step number three

is basically we're gonna take this gel

and we're gonna transfer it to a filter.

So, transfer the gel onto a filter.

And what the filter will basically allow us to do

is it allow us to visualize

'cause this gel is very flimsy.

So, we want to transfer it onto a filter.

What we'll do is we'll take a filter

that's basically the same size as the gel

and we're gonna basically just put it

right on top of the gel for a little bit

and the fragments will basically

transfer on to the filter.

So now, we're gonna have a filter with these fragments

and the filter is a lot sturdier than the gel.

So this is the filter and I'll just write

that down over here

and this over here is the gel.

Okay, so the next step, step number four

that we're gonna take the filter

and we're gonna expose it to a radio-labeled

the piece of DNA.

So, "expose to radio-labeled DNA."

Now, this radio-labeled DNA is going to be the complement

to our gene of interest.

So, we're interested in finding out

if Gene A is present in this mass of DNA over here.

So what we do is we're gonna take

the complementary sequence to Gene A

and radio-label it and expose it to this filter.

So, let's imagine that the radio-labeled

piece of DNA is this pink piece of DNA.

And let's imagine that we do have Gene A,

so let's imagine that this piece of this DNA fragment

was actually Gene A or our gene of interest.

So what's gonna happen is when we expose

the radio-labeled DNA to this filter paper,

it's going to anneal to our gene of interest.

So we're gonna have this radio-labeled

piece of DNA stuffed to this DNA fragment

which it's complement.

So, in order to visualize it,

in order to visualize this radio-labeled piece of DNA,

we have to do the fifth and final step

which is expose the filter to an x-ray film

in order to visualize the radio-labeled probe.

So, "expose to x-ray."

And the x-ray basically it will shoot a bunch of x-rays

and since this piece of DNA is radio-labeled,

it will pop up on the x-ray film.

So, we're gonna have a film and we'll draw

that film over here so we'll have this film

and basically the only thing that will pop up

is this fragment over here

and that fragment will have a control

and we'll be able to say,

"Okay. Well, since we have this fragment

"it's basically the radio-labeled piece"

"of DNA and since we see the radio-labeled DNA"

"it means that it had bound."

"It was bound to this Gene A"

"which means that Gene A was in this cup of DNA."