Probes and Blotting Methods

by Georgina Cornwall, PhD

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    00:01 How though do we end up seeing or using our DNA libraries? Once the DNA has been cloned, we can use it to probe for similar genes and other organisms. As you know, in order to get the process going, we are going to have restriction endonuclease breakdown our DNA.

    00:23 We will release our fragments of DNA and we are going to separate them using electrophoresis.

    00:31 Once we have our DNA in a gel, we need to denature the DNA so that we have single-stranded DNA stuck in the gel and now we want to ask in this DNA library that we cut up into thousands of fragments and run through a gel, do we have genes that we're particularly interested in? This is where we can start talking about different methods of visualization.

    00:59 In the last lecture, I introduced you to the idea that we could add luminescence to the DNA and visualize light on the DNA fragments but that to show us all of the DNA in the gel. We are looking now for specific sequences or we are probing for specific sequences.

    01:20 The technique is the Southern blot. It was named after the gentleman that first put the technique together, Dr. Southern. We will see that later on. There are Western blots and Northern blots but those guys weren't named Northern or Western at all. Different techniques, same principle. Southern blot involves DNA probes hybridizing with the DNA of interest to label it and show us if the sequences we're interested in or indeed in that DNA library. We have the electrophoresis gel. You will recall that the fragments are separated according to size as they move through the gels. Small ones move faster, longer ones are bigger have a harder time moving through the gel, so they move more slowly in order to get a visual of them. We are going to blot literally with a piece of blotting piece. We push it down onto the gel and that blotting paper will then have a copy of the DNA on it essentially.

    02:25 Now that we have that copy of DNA on our piece of blotter paper we need to hybridize it with some probes. Now you can order the probes these days. I am probing for this particular sequence or that particular sequence, look it up in a catalog, find it, have it delivered to you within a day or so and then you can go ahead and look for what you're looking for. This nitrocellulose membrane that was blotting is now in a sealed container because it has got radioactivity labelled DNA nucleotides in there and we allow those to hybridize. Let us take a look.

    03:05 Here we have a closeup of our gel and our bright green DNA probes looking for specific sequences.

    03:14 They float around in there and look for partner strands because we have denatured the original strand that was in there. Now we are going to ask the probe to bind to those and then we can wash off the gel, so that all the extra probes disappear and then visualize it. Hybridization of the radioactively labelled nucleic acids with the one settled in the gel will allow us to see it because we can now expose it to a film literally laying a photographic film over the top of this radioactive gel. We will create a print so that we can read the print and see the relative lengths of these pieces of DNA. And the labels that we have attached to them. We are looking specifically for these sequence and we see that there is one green spot on there and we know that length of a piece of DNA with that tag is associated with perhaps certain mutation that we are interested and then exploring in a certain type of cancer. So Southern blotting is a great technique for identifying DNA in a nitrocellulose gel. Northern, again this is not the guy's name.

    04:36 It's just sort of because we have Southern, why not we have Northern and Western. Northen blots are used to detect messenger RNA. Again we can use radioactive nucleotide probes. Western blotting, however, is used for proteins and in order to probe for proteins, we need to use antibodies that are radioactively labelled. The same thing proteins will move through a gel of electrophoresis gel. Larger ones move slower than smaller ones and then we can take a print of the radioactively labelled proteins of interest. Great techniques for visualizing what we have going on in gel electrophoresis as well as in perhaps DNA fingerprinting and such, which brings us right into the topic of DNA fingerprinting where we will get the

    About the Lecture

    The lecture Probes and Blotting Methods by Georgina Cornwall, PhD is from the course Biotechnology.

    Included Quiz Questions

    1. Southern blotting; TACCCGGTCGA
    2. Western blotting; TACCCGGTCGA
    3. Southern blotting; ATGGGCCAGCT
    4. Gel electrophoresis; DNA dye
    5. Gel electrophoresis; ATGGGCCAGCT
    1. Eastern blot — to study the defective proteins in a mixture of proteins
    2. Southern blot — detection of a specific DNA sequence in DNA sample
    3. Autoradiography — visualization of radioactive probe binding onto the molecule of question
    4. Northern blot — study of gene expression by detecting mRNA in the sample
    5. Western blot — identification of a specific protein in a protein mixture by using antibodies
    1. The size, its copy number and exact position of a cloned gene within the rDNA molecule
    2. The size, its copy number and exact position of mRNA onto the ribosome molecule
    3. The size, its copy number and exact position of tRNA onto the ribosome molecule
    4. The size, its copy number and exact position of rRNA onto the ribosome molecule
    5. The size, its copy number and exact position of mRNA, tRNA and rRNA molecules onto the ribosome molecule

    Author of lecture Probes and Blotting Methods

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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    By Bryson G. on 02. September 2020 for Probes and Blotting Methods

    She keeps it short and simple. Great work in this video. It's great prep before going into a lecture!

    By Eric C. on 22. July 2020 for Probes and Blotting Methods