Since you are putting in radiolabeled cDNA, does the DNA need to be separated before it is filtered?

Thank you for noticing this! I will add a clarification to the video to correct this omission.

Doesn't the radiolabeled DNA signal show up no matter what? How does its presence indicate the presence of the gene of interest? Is there a "wash" step I'm missing?

I managed to answer my own question on google. For anybody who reads this, yes, there is a wash step.

At 1:10, which enzymes can do this?

They are called restriction enzymes. They are enzymes discovered in bacteria that cleave DNA when it finds a particular nucleotide sequence (which are usually palindromic). These are used by bacteria as immune defence mechanisms against viruses.. There are hundreds of different DNA sequences that can be cleaved by different restriction enzymes, so if we know where on the DNA strand we want to cut, we can select a specific restriction enzyme to cut on or close to the place we want to break.

At 2 min 12 sec u told that dna would be separated based on size and BASED ON CHARGE. But the entire dna has same charge on it. How can charge play a role in separating ?

You are correct. After adding TAE or TBE to deprotonate the DNA, the DNA would be separated based on size alone, not by charge.

A previous question in this Biomolecules segment stated that if all molecules had the same charge they would not move regardless of the size.. I have always associated Gel Electrophoresis with size difference but now I'm just confused as to when each thing applies... can someone clarify ? thank you

for the sake of clarity, the question read like this "Electrophoretic separation at pH 6 of a sample of polypeptide 1 (mw 100) polypeptide 2 (mw 200) and polypeptide 3 (mw 400) would result in which of the following? (Note: the isoelectric point of each polypeptide occurs at pH 6)"

Would the radiolabled DNA be an antigen?

No, it's the complement of the gene we are trying to find.

How can you splice DNA so that you get whole gene fragments? It seems that the endonuclease could split the DNA in such a way that the gene of interest is fragmentized, keeping the probe from binding to the gene of interest. How can you keep this from happening?

I think you would cleave with restriction enzymes that are specific to sites of the strand that are on the outer edges of your gene of interest.

Does the process of enzyme digestion result in DNA cleavage? Is there a video I can watch that discusses this topic?

the video of restriction enzymes may be of help

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Southern blot

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Lauren
Posted 8 years ago. Direct link to Lauren's post “Since you are putting in ...”
Since you are putting in radiolabeled cDNA, does the DNA need to be separated before it is filtered?
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(11 votes)
Answer
- emilyabrash
  Posted 8 years ago. Direct link to emilyabrash's post “Thank you for noticing th...”
  Thank you for noticing this! I will add a clarification to the video to correct this omission.
  Comment on emilyabrash's post “Thank you for noticing th...”
  (2 votes)
kweiss14
Posted 3 years ago. Direct link to kweiss14's post “Doesn't the radiolabeled ...”
Doesn't the radiolabeled DNA signal show up no matter what? How does its presence indicate the presence of the gene of interest? Is there a "wash" step I'm missing?
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(6 votes)
Answer
- kweiss14
  Posted 3 years ago. Direct link to kweiss14's post “I managed to answer my ow...”
  I managed to answer my own question on google. For anybody who reads this, yes, there is a wash step.
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  (6 votes)
Upadhyayula Koushik
Posted 7 years ago. Direct link to Upadhyayula Koushik's post “At 2 min 12 sec u told th...”
At 2 min 12 sec u told that dna would be separated based on size and BASED ON CHARGE. But the entire dna has same charge on it. How can charge play a role in separating ?
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(2 votes)
Answer
- Kevin D. Fettel
  Posted 7 years ago. Direct link to Kevin D. Fettel's post “You are correct. After ad...”
  You are correct. After adding TAE or TBE to deprotonate the DNA, the DNA would be separated based on size alone, not by charge.
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  (6 votes)
studentn
Posted 6 years ago. Direct link to studentn's post “At 1:10, which enzymes ca...”
At
1:10
, which enzymes can do this?
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(3 votes)
Answer
- Liam.lah
  Posted 5 years ago. Direct link to Liam.lah's post “They are called restricti...”
  They are called restriction enzymes. They are enzymes discovered in bacteria that cleave DNA when it finds a particular nucleotide sequence (which are usually palindromic). These are used by bacteria as immune defence mechanisms against viruses.. There are hundreds of different DNA sequences that can be cleaved by different restriction enzymes, so if we know where on the DNA strand we want to cut, we can select a specific restriction enzyme to cut on or close to the place we want to break.
  Button navigates to signup page
  (3 votes)
Emily Bovasso
Posted 8 years ago. Direct link to Emily Bovasso's post “Would the radiolabled DNA...”
Would the radiolabled DNA be an antigen?
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(1 vote)
Answer
- Abid Ali
  Posted 8 years ago. Direct link to Abid Ali's post “No, it's the complement o...”
  No, it's the complement of the gene we are trying to find.
  Button navigates to signup page
  (3 votes)
christie2taylor
Posted 5 years ago. Direct link to christie2taylor's post “How can you splice DNA so...”
How can you splice DNA so that you get whole gene fragments? It seems that the endonuclease could split the DNA in such a way that the gene of interest is fragmentized, keeping the probe from binding to the gene of interest. How can you keep this from happening?
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(1 vote)
Answer
- Salma Soltani
  Posted 5 years ago. Direct link to Salma Soltani's post “I think you would cleave ...”
  I think you would cleave with restriction enzymes that are specific to sites of the strand that are on the outer edges of your gene of interest.
  Button navigates to signup page
  (5 votes)
Sharon Angarita
Posted 4 years ago. Direct link to Sharon Angarita's post “A previous question in th...”
A previous question in this Biomolecules segment stated that if all molecules had the same charge they would not move regardless of the size.. I have always associated Gel Electrophoresis with size difference but now I'm just confused as to when each thing applies... can someone clarify ? thank you
Button navigates to signup pageButton navigates to signup page
(2 votes)
Answer
- Sharon Angarita
  Posted 4 years ago. Direct link to Sharon Angarita's post “for the sake of clarity, ...”
  for the sake of clarity, the question read like this "Electrophoretic separation at pH 6 of a sample of polypeptide 1 (mw 100) polypeptide 2 (mw 200) and polypeptide 3 (mw 400) would result in which of the following?
  (Note: the isoelectric point of each polypeptide occurs at pH 6)"
  Comment on Sharon Angarita's post “for the sake of clarity, ...”
  (2 votes)
Julie
Posted 8 years ago. Direct link to Julie's post “Does the process of enzym...”
Does the process of enzyme digestion result in DNA cleavage? Is there a video I can watch that discusses this topic?
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(1 vote)
Answer
- Ms. MMDr
  Posted 8 years ago. Direct link to Ms. MMDr's post “the video of restriction ...”
  the video of restriction enzymes may be of help
  Button navigates to signup page
  (3 votes)
keanakickbooty
Posted 3 years ago. Direct link to keanakickbooty's post “At what point does the DN...”
At what point does the DNA become single-stranded? Or if it doesn't, how does the cDNA bind?
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(2 votes)
Answer
- Anatoly Rozhkov
  Posted 3 years ago. Direct link to Anatoly Rozhkov's post “The DNA strands get separ...”
  The DNA strands get separated after they have been cut by the restriction enzymes. The strands can be separated for example, by being exposed to high temperature, however I think there are other ways to separate DNA strands like incubating the DNA in some chemical solution (I couldn't find information on this, sorry).
  Button navigates to signup page
  (1 vote)
Mena
Posted 3 years ago. Direct link to Mena's post “why can the radio-labeled...”
why can the radio-labeled complementary gene radiate or be seen when only attach to the gene of interest?
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(2 votes)
Answer
- Anatoly Rozhkov
  Posted 3 years ago. Direct link to Anatoly Rozhkov's post “Radio-labeled DNA does no...”
  Radio-labeled DNA does not need to be hybridized to the complementary strand to emit radiation. The single-stranded unlabeled fragments are immobilized on the blot, and when the labeled DNA hybridizes it also becomes glued to the blot. The rest of the labeled (radioactive) fragments get washed away with a buffer.
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  (1 vote)

Video transcript

- 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."

MCAT

Course: MCAT > Unit 5

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Video transcript