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35 Cards in this Set
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Recombinant DNA is any DNA molecule made, that wouldn't normally exist in nature. Most common use is in gene cloning. |
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Primitive immune system of bacteria: Restriction enzymes are naturally occurring enzymes in bacteria. They will recognize and cleave specific sites within DNA. We have phages that will attach to the membrane/wall of the cell and inject its DNA genome into the cell, which will then be incorporated into the cells genome. So cells will need specific mechanisms to eliminate foreign DNA. Bacteria will methylate their DNA after cell replication in a way that's unique to that bacteria. If they spot unmethylated DNA, they will cleave and destroy this "foreign" DNA. The bacterial host is part of this primitive system (methylate and cleave DNA). It's going to have an enzyme that will look for the unmethylated DNA and create a double-stranded break at a specific site. For each bacteria, they are going to have a "restriction enzyme system" that recognizes a unique sequence to that bacteria. |
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The enzymes cut the DNA by grabbing it on both sides and cutting both strands of DNA at the same sequence. This is called leaving a "sticky" end or "overhang." The enzyme will cut in-between the A & G on the top and the A & G on the bottom. They are called sticky ends because they can ligate back together easily with the help of ligase to seal the nicks. We have different types of enzymes that will cut blunt ends to leave a "clean" break that won't make a sticky end. |
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Here we are cutting in-between the A & G on both sides. Restriction enzymes that cut blunt ends will also recognize palindromic sequences, but will just cut straight through and not at an angle. |
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C. The answer is not supposed to be the same forwards and backwards, so that eliminates A, B & D. |
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D. It will always leave a 3' Sticky end if the cut is to the far right. It will always leave a 5' sticky end if the cut is to the far left. |
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A molecular clone is a gene that we are replicating in a bacterial cell. We can do this by taking a gene and splicing it into vectors. This is a Standard Cloning Vector. 4.7kb in length. We have restriction enzyme sites. 1 for EcoRI, 2 for HindiIII etc. We also have an origin of replication. An origin of replication ensures that a bacteria will be replicating a copy of the plasmid in it. We splice in a gene we want the bacteria to turn into a protein. We do this by cutting our gene product and plasmid with the same RE. This will create complementary sites for both ends, so the sticky ends from the plasmid can match up with the sticky ends with the gene we want. Not all plasmids will have a RE site that will be the same as the gene product, so it doesn't work sometimes. |
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One of the ways we overcome the problem of not have all the same RE sites on one plasmid. We can engineer plasmids to have a Polylinker, which is a sequence of DNA we've synthesized that contains a sequence a lot of RE will recognize. By doing this, we increase the diversity of cloning experiments. Other components of vector: In addition to OOR that allows us to replicate this and pass it on to daughter cells, we have different promoter and operator sequences. We also have a Ribosome binding site ( AKA Shine Delgarno Sequence). The operator and promoter sites are just upstream of the SDS. The polylinker is the site where we clone in our gene (MCS). This is where we have our RE sites. We'll have many different RE sites to choose from but they will have to be close to flanking the 3' and 5' regions of our coding protein. We can have the plasmids be inducible, which means they can be turned on only in the presence of a certain molecule or factor. This figure shows that we have 2 different populations of bacterial clones that are identical. They each have a copy of a plasmid with the gene RecA in the Polylinker. They can both express RecA, but in one instance we get an effector molecule that has an influence to turn on the level of expression by exposing the bacteria to an effector. You can take the regulatory sequences that control the lac operon, introduce them here and regulate the expression of RecA (in place of lacz, laco etc) by the presence of allolactose. So only make RecA in presence of allolactose. So take the regulatory sequence of one thing and introduce to the coding region of a plasmid. |
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.We have to cut the part we will be cloning into the plasmid with the same RE as the plasmid itself. This will create complementary sticky ends, which will allow the gene to ligate into the plasmid. This won't happen all the time because the cut sequences are highly complementary to each other and can just re-attach. When this happens, we call it an empty vector. To decrease the chances of this happening, we just increase the amount of product we insert in for a higher chance that it will attach to the plasmid (vector). |
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This shows a vector with 4 different polylinker sites. All these sequences are what the enzymes recognize. The sequences overlap so we can end up with a very short polylinker but that will actually be able to have many enzymes cut it. Polylinkers can be incorporated into plasmids, they just need to be cleaved by the same enzyme. |
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Can have even bigger polylinkers |
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B. There are 2 BamHI sites |
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We can mass produce insulin through bacteria |
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Insulin is produced from bacteria. Bacteria don't have a natural insulin gene, so we have to introduce the human form of insulin gene into the bacteria. |
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We do this by taking the human insulin gene and cloning it into an expression vector. We have our bacterial plasmid (red) and we cut that with the same RE as we do our gene of interest. We ligate these together and force the bacteria to take up that plasmid to use it. Once we grow these bacteria to produce the insulin, we can isolate and purify the insulin grown from bacteria in large quantities. |
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We'll want to join to molecules together. We cut the insert and vector with the same RE. they have complementary sticky ends which can seal together. We will need DNA ligase to seal them together. |
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Not all ligation reactions will work. If we have something cut a sticky end and the other with a blunt end, they will not join. You can sometimes get non sticky ends to ligate if they aren't entirely complementary. |
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Thinking back to PCR, we design primers that are flanking either upstream/downstream our gene of interest. When we design primers, the best and easiest way to do it is to have primer sequence include the RE site we want to use. |
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What the plasmid will look like once we've ligated everything in. We have our human insulin gene located just downstream a promoter region. This expression vector can be used by bacteria to synthesize/transcribe human insulin gene and translate it into a protein. |
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One thing we have to consider is that bacteria don't have splicing machinery. It won't do us any good to insert genes with introns. We have to take pre-spliced regions of DNA and insert them. We do this by making a cDNA of mRNAs made by a cell (made by Euk). Because all cDNAs have a poly-A Tail, we can use that to isolate them, and reverse transcribe them into mRNA. Once we've isolated all mRNA, we can use an OligoDT primer to bind the PolyA Tail, use reverse transcriptase to use the mRNA as a template to synthesize a CDNA complement. We use RNAse to degrade the other half of the RNA and use random hexamers to fill in the opposite sequence. |
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We can use this to make a library of all the genes that are being expressed in a cell. A cDNA library takes all the mRNA from a cell, converting them into a double stranded cDNA. Each cDNA will go into it's own vector. cDNA library is representative of what was being originally transcribed in that cell. Has only the sequence we need to create protein. Have multiple copies of each cDNA if the original gene has high expression. Genomic library takes the whole genome, and doing a light digestion of DNA to break into small fragments. Each fragment can be ligated into it's own plasmid. Has introns, promoter regions, long repetitive sequences etc. Use this library if we want to study the source of DNA or if we want to study what is present in DNA. Has only 1 copy of each sequence. |
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Contains DNA copies of all mRNAs. We can use the sequences, put them in plasmids and bacteria can use that to transcribe and make RNA/Protein. Eliminates the step of splicing introns. Because only a small part of our genome is protein coding, we won't have the intervening, repetitive, intron sequences. |
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If we have a cell undergoing mitosis, these will have identical copies of DNA. Different cells will express different genes, like nerve cells and skin cells. So if we were to take the cell itself, the genomic library would be the same, but the cDNA library would be different because every cell will have the same genomic DNA, but will have a different cDNA library because they will express different genes. So cDNA libraries are useful to see what gene is expressed the most between 2 cells. |
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Have a polyA Tail, which we can hybridize an OligoDT primer to. We can extend this primer to get a double stranded molecule which will have half RNA and half DNA. We can use RNAse to get rid of remaining RNA and we can build up the complementary sequence to then end up with double stranded cDNA. This is what we would be cloning into a vector. |
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Genomic library has many different types of sequences. We can use enzymes to break up each sequence, and we can use these many different sequences to go into their own plasmid. |
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A. Will be genomic if it's anything that's not part of the mRNA; Intron, promoter sequence or anything that's not transcribed or translated |
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Isolate all the mRNA of a specific human cell. The mRNA for something like an insulin gene will be here in the sample. We make a cDNA library of this, and a cDNA copy of the mRNA that was there. Now we have a mixed population. To make sure our gene of interest is inserted into the plasmid, we use primers that are specific for our insulin gene. Those are going to amplify only the insulin gene from our cDNA library. At the end of the PCR cycle, the vast majority of the genes will be our insulin gene. Then we can cut because our primers have a RE site. We can then treat our product with ecoRI and our plasmid with ecoRI, which will allow both to ligate to each other. |
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No we've made the plasmid that we want and we need to get it inside the cell by a process called transformation. We take a recombinant DNA plasmid and introduce into a host cell. |
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The two main methods are to treat the cells with calcium chloride which will help make pores in cell wall which will allow plasmid to slip through. We can also electroporate cells, which will cause transient breaks in the membrane. If we have a ton of the DNA plasmid, the plasmid can get in through one of these breaks. |
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Sometimes after all of this, only one of the bacteria has the plasmid. We use some sort of selectable marker that is on the plasmid and follows wherever the gene is going. It gives the bacteria that is transformed with the plasmid another ability (glow, be resistant to a certain antibiotic etc). This is a way to isolate the bacteria we need. |
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If we had a group of cells that were grown on tetracycline, this only ones that would survive are the ones with the plasmid we want. |
