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Animation: Creating Recombinant DNA

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Hey there, this is Eric Simon from New England College in Henniker, New Hampshire. In this Visualizing the Concept video we'll walk through the process of creating recombinant DNA. Let's get started. Many genetic engineering experiments involve combining DNA from more than one source often from different species. For example, a human gene encoding for an important protein may be inserted into a bacterium. Such a modified bacterium will then reproduce and express the human protein which can then be harvested in large quantities perhaps by a pharmaceutical company to produce a new drug. In another example, an insect gene might be placed into a plant in order to improve that plant's ability to resist cold weather. To create recombinant DNA scientists must cut pieces of DNA from multiple sources and then past them together into a single recombinant DNA molecule. In this animation we're going to focus on the actual cutting and pasting of DNA to see how a typical genetic engineering project is undertaken. To help visualize this process let's use household materials. Imagine that you have a necklace made from blue ribbon and you wish to insert a bit of red ribbon into it. To do this you would snip out a piece of the red ribbon by making two cuts, then open up the blue necklace by making one cut. You could then insert the new ribbon and seal the gaps. You now have a recombinant necklace that contains pieces from two different ribbons combined together. To accomplish a similar process with DNA genetic engineers often start with plasmids, small circular pieces of DNA found within many bacteria such as this E. coli. Once a researcher has removed a plasmid from a bacterium it can be manipulated in the laboratory. In this experiment the circular plasmid needs to be cut open. This is accomplished using a restriction enzyme, a protein that cuts DNA at one specific sequence of bases. The restriction enzyme shown here for example is called EcoR1. It cuts DNA at one specific sequence of bases called the restriction site. For example, the restriction enzyme EcoR1 will only cut DNA at the sequence G-A-A-T-T-C. When this restriction enzyme is added to the plasma DNA it will cut the DNA at every location that contains the sequence and no places that don't. The specific restriction enzyme used in the genetic engineering project varies from experiment to experiment, but an engineer will usually choose a restriction enzyme that will cut the bacterial plasmid in just one location opening up the circle. If a plasmid contains two restriction sites for a particular restriction enzyme how many separate pieces of DNA will result? The result of cutting a circular plasmid twice will be two linear pieces of DNA. Imagine snipping a rubber band twice, you'll be left with two pieces. Once the bacterial plasmid is snipped open it's ready to have another piece of DNA inserted into it. Here you can see a human cell that contains a target gene. Once the DNA is extracted from the cell the same restriction enzyme can be used to cut it up. Because the human chromosome is so big it will probably be cut into several pieces, one of which contains our target gene shown in red. If a linear piece of DNA contains two restriction sites for a particular restriction enzyme how many separate pieces of DNA will result? In this case since the starting DNA is linear two cuts will result in three linear pieces of DNA. Imagine cutting a strip of paper twice, you'd be left holding three smaller strips of paper. Now that we have our two desired segments of DNA, one bacterial plasmid and one human gene, let's see how they're pasted together. The key is that the restriction enzyme used did not cut across the DNA double helix evenly. Instead it left staggered cuts called sticky ends. Each sticky end is a single stranded set of unpaired DNA bases. The bases in the single stranded regions are no longer hydrogen bonded to their matching bases on the other strand. These unpaired bases are now available to pair up with a complementary set of bases on another sticky end. Notice that the DNA base sequences of the sticky ends on the bacterial plasmid DNA and the human DNA match up. The key to creating recombinant DNA is to use the same restriction enzyme to cut both the plasmid DNA and the target DNA. Because the same enzyme was used the same sequences were cut in the same way. As a consequence the unpaired based sequences are complementary to one another and the sticky end of one piece of DNA will match up with the sticky end from another. The hydrogen bonds that form between the sticky ends of the two pieces of DNA temporarily hold the foreign DNA in the middle of the plasmid. To permanently pasted these separate pieces of DNA the engineer can add a DNA pasting enzyme called DNA ligase. This enzyme builds new covalent bonds sealing up the separate pieces of DNA into a single recombinant piece of DNA. The goal of creating recombinant DNA has now been achieved. Once created, the recombinant plasmid can be inserted into a bacterium. The recombinant plasmid will multiply to produce a colony of identical bacteria, each of which carries the foreign gene in the recombinant plasmid. As it goes about its life cycle a recombinant bacterium will treat the foreign DNA as if it were its own. This means that the foreign DNA will be transcribed into an RNA molecule and then translated into a protein. Perhaps the protein is a pharmaceutical product that can serve as a drug. Right now in the U.S. Mid-West there are large tanks of recombinant bacteria carrying the gene for human insulin. The bacteria transcribe and translate the gene to produce human insulin protein which can then be extracted and purified. This human insulin is then packaged and sold to millions of diabetics for whom this drug made from recombinant DNA is a life-saving medication.
Hey there, this is Eric Simon from New England College in Henniker, New Hampshire. In this Visualizing the Concept video we'll walk through the process of creating recombinant DNA. Let's get started. Many genetic engineering experiments involve combining DNA from more than one source often from different species. For example, a human gene encoding for an important protein may be inserted into a bacterium. Such a modified bacterium will then reproduce and express the human protein which can then be harvested in large quantities perhaps by a pharmaceutical company to produce a new drug. In another example, an insect gene might be placed into a plant in order to improve that plant's ability to resist cold weather. To create recombinant DNA scientists must cut pieces of DNA from multiple sources and then past them together into a single recombinant DNA molecule. In this animation we're going to focus on the actual cutting and pasting of DNA to see how a typical genetic engineering project is undertaken. To help visualize this process let's use household materials. Imagine that you have a necklace made from blue ribbon and you wish to insert a bit of red ribbon into it. To do this you would snip out a piece of the red ribbon by making two cuts, then open up the blue necklace by making one cut. You could then insert the new ribbon and seal the gaps. You now have a recombinant necklace that contains pieces from two different ribbons combined together. To accomplish a similar process with DNA genetic engineers often start with plasmids, small circular pieces of DNA found within many bacteria such as this E. coli. Once a researcher has removed a plasmid from a bacterium it can be manipulated in the laboratory. In this experiment the circular plasmid needs to be cut open. This is accomplished using a restriction enzyme, a protein that cuts DNA at one specific sequence of bases. The restriction enzyme shown here for example is called EcoR1. It cuts DNA at one specific sequence of bases called the restriction site. For example, the restriction enzyme EcoR1 will only cut DNA at the sequence G-A-A-T-T-C. When this restriction enzyme is added to the plasma DNA it will cut the DNA at every location that contains the sequence and no places that don't. The specific restriction enzyme used in the genetic engineering project varies from experiment to experiment, but an engineer will usually choose a restriction enzyme that will cut the bacterial plasmid in just one location opening up the circle. If a plasmid contains two restriction sites for a particular restriction enzyme how many separate pieces of DNA will result? The result of cutting a circular plasmid twice will be two linear pieces of DNA. Imagine snipping a rubber band twice, you'll be left with two pieces. Once the bacterial plasmid is snipped open it's ready to have another piece of DNA inserted into it. Here you can see a human cell that contains a target gene. Once the DNA is extracted from the cell the same restriction enzyme can be used to cut it up. Because the human chromosome is so big it will probably be cut into several pieces, one of which contains our target gene shown in red. If a linear piece of DNA contains two restriction sites for a particular restriction enzyme how many separate pieces of DNA will result? In this case since the starting DNA is linear two cuts will result in three linear pieces of DNA. Imagine cutting a strip of paper twice, you'd be left holding three smaller strips of paper. Now that we have our two desired segments of DNA, one bacterial plasmid and one human gene, let's see how they're pasted together. The key is that the restriction enzyme used did not cut across the DNA double helix evenly. Instead it left staggered cuts called sticky ends. Each sticky end is a single stranded set of unpaired DNA bases. The bases in the single stranded regions are no longer hydrogen bonded to their matching bases on the other strand. These unpaired bases are now available to pair up with a complementary set of bases on another sticky end. Notice that the DNA base sequences of the sticky ends on the bacterial plasmid DNA and the human DNA match up. The key to creating recombinant DNA is to use the same restriction enzyme to cut both the plasmid DNA and the target DNA. Because the same enzyme was used the same sequences were cut in the same way. As a consequence the unpaired based sequences are complementary to one another and the sticky end of one piece of DNA will match up with the sticky end from another. The hydrogen bonds that form between the sticky ends of the two pieces of DNA temporarily hold the foreign DNA in the middle of the plasmid. To permanently pasted these separate pieces of DNA the engineer can add a DNA pasting enzyme called DNA ligase. This enzyme builds new covalent bonds sealing up the separate pieces of DNA into a single recombinant piece of DNA. The goal of creating recombinant DNA has now been achieved. Once created, the recombinant plasmid can be inserted into a bacterium. The recombinant plasmid will multiply to produce a colony of identical bacteria, each of which carries the foreign gene in the recombinant plasmid. As it goes about its life cycle a recombinant bacterium will treat the foreign DNA as if it were its own. This means that the foreign DNA will be transcribed into an RNA molecule and then translated into a protein. Perhaps the protein is a pharmaceutical product that can serve as a drug. Right now in the U.S. Mid-West there are large tanks of recombinant bacteria carrying the gene for human insulin. The bacteria transcribe and translate the gene to produce human insulin protein which can then be extracted and purified. This human insulin is then packaged and sold to millions of diabetics for whom this drug made from recombinant DNA is a life-saving medication.