Professor: This video or this powerpoint is going to look at recombinant DNA.

Recombinant DNA is the general term for taking a piece of DNA and combining it with another trand of DNAs. You’re taking it from one organism and you are inserting it to the DNA of another organism. By combining two or more different strands of DNA, scientist are able to create a new strand of DNA. In most cases, recombinant DNA is formed when DNA fragments from two or more different organisms are spliced together in a laboratory.

And we are going to look at now how we splice or how we put together different parts of DNA. So, in this technique, DNA is cut at specific sites by a restrictive enzyme that locates a specific sequence of amino acids within the DNA and cuts it at that location, leaving DNA fragments with unpaired nitrogenous bases or “sticky end”.

So what this is saying is, we take an enzyme, and we take an enzyme that we know exactly where it cuts DNA at. So you might have an enzyme that cuts DNA, always wanna find an A-A-T-T. So everywhere it find an A-A-T-T, it’s going to make a cut there, and it’s going to separate those bases apart from their base pairs. And it will always cut at that point. So it’s useful that way because we know that when we stick those enzyme there with the whole wack of DNA, when things got broken apart, we know exactly where it has been broken apart in every single strand of DNA. We know that it’s been, as my example goes, that it has been broken apart in that A-A-T-T. Everywhere along the line. So we know that exactly. And different enzyme is going to break apart DNA at different parts, at different amino acid strand.

So what this mean is that you’ll get a situation where…if this is an enzyme like I said separate and enzyme(points to ppt), everywhere it finds the A-A-T-T. And the pair of that, obviously if we look at the other side of that, that’s matching up with T-T-A-A. If the enzyme, always separate at A-A-T-T, everywhere that it finds it along that strand, it’s gonna separate it. And it may find A-A-T-T a couple of times along the here, and it will separate that A-A-T-T, and it will break it apart again.

So what we do is we take an enzyme and we’ll stick in it with what’s called the plasmid. This is a fraction of DNA from a bacteria and we know that it’s going to break it apart at A-A-T-T.

And when we put that enzyme in with say, a piece of animal DNA, we know that it also gonna cut the animal DNA at A-A-T-T. And as you go along here, you’ll see that “oh, there’s another A-A-T-T” so it cuts that, and there’s another A-A-T-T. And everywhere you find that sequence of amino acid, it’s going to make a break. It is going to break that DNA at that point.

Now, inside bacterial cell there is only one single strand of DNA. Bacteria doesn’t have 23 pairs of chromosomes like humans do. Bacteria has this, single strand of DNA like this. And it’s in a circle like that. Sometimes, however, there are other smaller rings of DNA that are found in bacteria as well. These smaller rings are called plasmids. (points pictures on powerpoint).

Plasmid is just a little piece of DNA. It doesn’t obviously contain as much information as the DNA of the bacteria does but it contains some useful things that bacteria can use for information. Plasmids are not essential for the bacteria, it can survive without it, but they may help them to survive in some threatening environment.

So what plasmids do is they contain information about how to may be fight off antibiotics or how to survive in an environment with antibiotics or they contain information about how to survive in harsh environment like the really salty environment. Or they might contain information on how to live in thermal vents, like those areas underneath the ocean, near the ocean floor… very very hot. So the plasmids are essential in the sense that they carry the protein and the enzyme that the bacteria needs to survive in a normal environment. But they contain some interesting information that might help them in harsh environment.

So when it says that we’re taking up a plasmid, that little section of it from a bacteria. (And this is the same diagram as before.) Remember that enzyme is cutting at A-A-T-T.

So the function of plasmid within a bacteria is often to produce proteins that protect the bacterium from harmful substances such as antibiotics, or if there’s heavy metals in the area or excess nutrients. Things like that. So it’s kind a like this special powers that gives it. It’s not necessarily essential but helps when the environment gets threatened.

By treating plasmids with the same restrictive enzyme as the DNA molecule that you wish to make in you recombinant DNA, the plasmid DNA is cut at exactly the same location with these several sticky ends. What that means is, as I said before, we take the enzyme, we stick it over the plasmid and then cut it at A-A-T-T. So now when we stick it with animal DNA for example, it also cuts it at A-A-T-T. And that leaves the same ends. (points example in powerpoint)

So one fragment of DNA from an organism is inserted into the plasmid DNA where bonding occurs between complementary nitrogenous bases creating what we called, recombinant DNA. The bonding of two DNA sections is made possible or facilitated by DNA ligase which connects the sticky ends together.

So you’ve got A-A-T-T that’s available here, open on this side, and T-T-A-A is open on this side. And what we do is take this plasmid and we stick it in a piece of plasmid, say the animal DNA. The ends are open, and so, this ends is gonna matched up with this ends. And they do that when we put what is called DNA ligase. So that’s the enzyme that is going to bond it together, it glues them together. And they’re able to glue together because the same enzyme has cut this at exactly the same spot as it is cut in animal DNA. So the same ends are open. So we can take A-A-T-T from here and that will bind with  T-T-A-A from here. And we call it sticky ends because they are able to bind together with their base pairs on the other side. Ok? and so again, this is all happening  we stickin an enzyme called DNA ligase. The ligase goes along and it connects, it glues them back together like there.

So this type of technology allows us to take a gene from one organism and insert it into the DNA of another organism. This allows the new DNA strand to produce a protein it previously could not produce. An example of this is taking a gene that codes for insulin production and we need that, we use insulin for regulating or blood sugar levels. And inserting it to a bacterial DNA. Now, the bacterium DNA will be able to produce insulin proteins which can be used by humans to treat diabetes.

And this is an example of insulin, an insulin crystal that was used or that was created by recombinant DNA and bacteria. Now what that means is, we take the gene from our DNA that says, “this is how you make insulin”. And we take that DNA, that gene from our DNA, and we insert it to the bacterial DNA. So if you look at it, we don’t have that gene before. Well we break it apart, we break our DNA apart add that section that codes for that protein that tells our body how to make insulin. An we take that insulin gene and we insert it, and we stick it into the bacterial DNA. Now what it says in here is, “make this bacterial protein, make this bacterial protein, make this bacterial protein, and oh! make insulin”. And it didn’t have that insulin before but it is able to make insulin now.

What we can do is we can culture millions and millions and millions of cells of bacteria that now sure to make insulin. And it’s the same insulin that we use because it is our gene that we gave that bacteria. So it’s going to make the insulin that we use in our bodies.

So why is recombinant DNA important? We are now able to use recombinant DNA to produce:

• Human growth hormone, which we were not able to do before in vast amounts. It gets easier when you got bacteria to do it.
• Erythropoietin which is responsible for the production of red blood cells (that’s very important to people)
• Better crops, that are resistant to pests, herbicides, and pesticides

And essentially what recombinant DNA has allowed us to do is we can look at the genes of any organisms and if we find that we think might be useful to another organism, we can just insert it in there. And so we get things like tomatoes that have animal genes in there where the shell life is longer and you’ll have things like crops that we take genes from other organisms that allow them to be resistant to different pesticides and chemicals or resistant pests in general. So we are able to take pieces DNA from all sorts of organisms and we can put them into other organisms and we are basically what we are tinkering what the DNA that’s in this organism, we are creating this designer organisms that are able to function the way that we want them to function.

Leave a Reply