Enviropig Step 4

Dept. of Agronomy and Horticulture Author

01/26/2016 Added

574 Plays

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In step four of Enviropig we learn about how DNA is analyzed to ensure that the transgene has been incorporated into the transgenic organism. For more information visit the Enviropig website: https://ge.unl.edu/enviropig/

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[00:00:07.188]To start the process of DNA analysis,
[00:00:08.988]you first need to collect tissue samples
[00:00:10.388]from the pigs in our breeding program.
[00:00:12.228]We will use tail clippings
[00:00:13.868]since pigs tend to chew each other's tails.
[00:00:15.868]We will have to clip them anyway,
[00:00:17.468]so it's convenient to use this tissue.
[00:00:19.688]We could also obtain samples from ear notches
[00:00:21.856]or collect cells from any other part of the pig
[00:00:23.796]since the DNA is the same in every cell.
[00:00:25.976]We need to extract the DNA in order to see which of the pigs
[00:00:28.897]carries the trans gene.
[00:00:30.356]Pigs that do carry the trans gene
[00:00:32.316]will be used to make future crosses in the breeding program.
[00:00:35.376]To start, I'll add some lysis buffer to each sample.
[00:00:37.876]This breaks open the cell membranes,
[00:00:39.756]which explodes the cells and releases the DNA into solution.
[00:00:42.916]We'll then add some protease
[00:00:44.616]to break down the proteins in the solution.
[00:00:48.216]So now I'm gonna load these up into the centrifuge.
[00:00:52.204]And now I'm going to run this
[00:00:54.305]at the highest speed possible for about five minutes
[00:00:56.785]and that should be enough to separate
[00:00:57.985]all the solid components down to the bottom
[00:01:00.085]and just leave that liquid with the DNA on top.
[00:01:06.245]It's a little difficult to see on the side,
[00:01:07.725]but you can tell at the bottom there,
[00:01:09.186]we have a nice pellet of those solids forms
[00:01:12.725]and on top we have that elute
[00:01:14.685]or the liquid with our DNA and other contaminants in it.
[00:01:19.065]So, that's what we're gonna go purify
[00:01:20.905]and extract our DNA from now.
[00:01:24.245]So right now I'm adding isopropanol, also known as,
[00:01:26.885]rubbing alcohol to the lysate
[00:01:29.465]and that's gonna help us precipitate out the DNA
[00:01:31.525]just so it's a little bit more accessible
[00:01:33.165]for our magnetic beads.
[00:01:35.785]Next I'm gonna add MagAttract beads
[00:01:37.225]and this is actually going to attach to the DNA.
[00:01:39.225]And then we can use these basically as a handle
[00:01:41.825]to move the DNA around once we actually have it
[00:01:43.645]in our extraction device.
[00:01:45.785]The device I have here is called the BioSprint
[00:01:47.705]and basically it's just going to do all the work
[00:01:50.245]of actually transferring those magnetic beads
[00:01:52.305]from wash to wash for us.
[00:01:53.945]Just making the extraction a little bit more simple.
[00:02:01.045]I have here our plate,
[00:02:02.111]it's prepared for a PCR reaction
[00:02:04.552]and it has our genomic DNA
[00:02:06.791]and then just all the raw components we need
[00:02:08.571]just to amplify a really small section of that DNA.
[00:02:12.011]We design what are called primers,
[00:02:14.591]which are basically small molecules
[00:02:15.951]that will pinpoint only certain parts of the DNA.
[00:02:18.651]And so we design these primers to target
[00:02:20.732]around our trans gene,
[00:02:22.591]or basically fingerprint our trans gene.
[00:02:24.552]And so we want to see amplification of just that region.
[00:02:27.871]So I'm gonna go ahead and put the plate
[00:02:29.511]into our thermocycler.
[00:02:31.691]And we're gonna go ahead and run our program.
[00:02:33.231]This is gonna cycle our temperature for us
[00:02:35.131]and will make all the copies of our trans gene
[00:02:38.811]that we're interested in.
[00:02:40.311]After we have our copies made,
[00:02:41.751]we're gonna take that over to our gel boxes
[00:02:44.271]and run a gel electrophoresis to see
[00:02:46.111]whether that gene is present or absent.
[00:02:48.851]So in order to actually visualize the bands in the gel
[00:02:51.771]we need to have millions or even billions of copies
[00:02:54.691]of the segment of DNA we're interested in
[00:02:56.831]and that's why PCR is so powerful to us.
[00:02:58.991]It enables us to actually observe
[00:03:03.211]changes in the DNA at eye level
[00:03:06.190]which is an amazingly powerful tool
[00:03:07.750]for breeders and scientists alike.
[00:03:10.570]We might be looking to see if we have our transgenic gene
[00:03:13.830]in each of these wells.
[00:03:15.710]We're gonna do is take this,
[00:03:17.470]load it into our gel
[00:03:19.210]and after running it
[00:03:22.590]we'll be able to see whether or not
[00:03:24.210]a band is present or not.
[00:03:25.490]If a band is present,
[00:03:26.570]it means there's amplification
[00:03:27.890]and the gene is present.
[00:03:29.290]If there's no band, then we can assume
[00:03:31.790]that the gene was not successfully transferred
[00:03:33.950]into the plan.
[00:03:35.590]So because the DNA backbone is negatively charged,
[00:03:38.510]when we place it in an electric field,
[00:03:40.750]it's going to be attracted to the cathode,
[00:03:43.210]or the positive end of that electric field
[00:03:45.390]and so we load our DNA over here
[00:03:48.430]on the left side of a given comb in a well.
[00:03:51.410]And our positive charge is over here.
[00:03:54.590]So once we turn our gel box on,
[00:03:56.430]our PCR product, our DNA fragments
[00:03:59.450]are going to be attracted to this cathode
[00:04:01.890]and they're gonna be drawn through the gel this way.
[00:04:04.230]And our gel is full of tiny pores
[00:04:06.710]and holes so it's gonna act as a seed for us.
[00:04:11.070]So smaller bands, smaller PCR products
[00:04:14.350]are gonna be able to move through these holes
[00:04:15.790]much more rapidly than say larger bands.
[00:04:18.530]So that'll help us actually separate out band sizes.
[00:04:22.170]So if you let this gel run for say six hours,
[00:04:24.730]I can tell the difference between PCR product
[00:04:27.590]with say 250 base pairs and one with 260.
[00:04:34.590]So I have here a gel,
[00:04:35.950]it's been ran in electrophoresis for about six hours
[00:04:39.470]and it's just been stained with a chemical
[00:04:42.630]that absorbs UV light.
[00:04:44.670]And so we're gonna do is place it in this device here,
[00:04:47.850]it's basically a UV camera on top
[00:04:50.850]and just a giant UV-emitting panel on the bottom,
[00:04:55.210]just kinda like a tanning bed.
[00:04:56.770]And what I'm gonna do is place the gel in here.
[00:05:05.370]Perfect, so our PCR was successful.
[00:05:07.570]We can see that.
[00:05:09.010]So these presence-absence tests basically tell us
[00:05:11.510]whether or not the trans gene that we're interested in
[00:05:14.470]is actually present.
[00:05:15.530]And this helps us out and helps our breeders out
[00:05:18.130]because we can tell them
[00:05:18.910]which lines to continue investing time and resources in
[00:05:21.630]and which lines to give up.

Enviropig Step 4

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