Lecture on Immunology 1
Immunology is the science of body's defence system. This video presents MIT lecture on immunology. The lecture was delivered by Professor Robert A. Weinberg. Edited by Ashraf. .
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Added: April 2, 2009, 4:51 am
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Cell Cycle/Signaling Lecture
Biology > Introduction to Biology/nVideo Lectures - Lecture 19/nTopics covered: /nCell Cycle/Signaling/nInstructor: /nProf. Robert A. Weinberg
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Added: April 2, 2009, 4:46 am
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Recombinant DNA Lecture 3
Biology > Introduction to Biology/nVideo Lectures - Lecture 17/nTopics covered: /nRecombinant DNA 3/nInstructor: /nProf. Eric Lander/n /nTranscript - Lecture 17/nGood morning. Good morning. I don't know about you, but I can't take too many more nights like this. I confess, I haven't gotten a thing done for so many nights in a row now, but what a game! How many of you saw the game? Excellent. Very good, very good. You have your priorities straight in the world. Very good./nWell, if it's possible to get your minds off Curt Schilling last night, and off more importantly tonight. Perhaps we can spend a bit of time this morning in the meanwhile with whatever spare neurons you have talking about recombinant DNA for a bit, OK? What we talked about last time was different ways to clone your gene based on its properties. We started off with cloning by complementation, right, the idea that if you took a library of clones, you would be able to put it into bacteria and select a bacterium whose phenotype had been restored by virtue of having the plasmid./nYou would complement the defect. You'd find the clone you wanted because it complemented the defect. That's great if you can put it into an organism that has a defect. You can do it with bacteria. You can do that with yeast. It's harder to do with large organisms because you can't inject enough of them with different clones to be able to make that practical unless you're working in cell culture or some very small, fast growing organism./nWe talked about being able to use a protein sequence, reverse translating that protein sequence in the computer from amino acid sequence to nucleotide sequence, and using the nucleotide sequence to design a probe to hybridize back to the genome. That works fine if you have a protein sequence./nBut the last topic we talked about that I wanted to just touch on again this morning was suppose you were trying to clone the gene that causes a certain human disease, and you have no idea what the protein was. Then, you can't use its amino acid sequence because you don't have the protein. What can you possibly do when all you know is that you have a gene which causes a genetic defect that causes a disease? And I said you could clone it using the ideas of genetic mapping, position, the things that Sturtevant developed. And, I touched on it briefly, and I want to just touch on it a bit more because some people had some questions about it./nAnd I've set up a very simple example to show you. Suppose that, to make it easy, we're working in a fruit fly first. We're working drosophila, and suppose that the true picture of the underlying chromosome is like this. There's a locus that could either have a mutant allele M or the wild type allele plus. There's a bunch of other loci along the chromosome. And, let's suppose we know all of where they are and all that. And, they have two alternative alleles./nAt this locus the alleles are orange or pink. At this locus I'll call the alleles orange or pink. Now, these are different loci. These are different alleles. I've just called them orange and pink in both cases so I don't have a rainbow of colors up here to confuse us. But all I mean is there's two possible alleles here, two alleles here, two alleles here. This is the diseased gene we're interested in, and these are passive markers. These are other markers along the chromosome./nIf we were to set up a cross between heterozygotes, a heterozygote here, and a heterozygote here, and it were the case that on the chromosome bearing the mutant allele, it happened that at these three markers we had orange alleles. I don't know what they are, but whatever these orange alleles are, they might be a visible phenotype, forked or yellow or bristled. They could be a DNA sequence difference. They could be whatever you want, but let's suppose the M chromosome has a set of alleles that are different in each location than the plus chromosome./nThen, when we look at the offspring that come out of this cross, let's only, for the sake of simplicity, look at those offspring who are homozygous mutants. Well, in general, if there's been no crossover here, then the M chromosome will have orange, orange, orange, orange, orange, orange. If there's been a crossover, however, it could go orange, orange, pink on one of those chromosomes. Or if there's been crossovers like this, it could go orange, orange, pink on one chromosome, and orange, pink, pink on the other chromosome./nIt could even, in the extreme, have had crossovers very close to the gene maybe here, and even maybe here. And you've got orange, pink, pink, and pink, pink, pink. But if we look at the many segregates, you know from genetic mapping that the closer the locus is to the disease gene, the more strongly correlated the inheritance will be, the tighter the linkage will be. This is nothing more than linkage mapping./nBut now, suppose we were doing linkage mapping, but for the sake of argument the whole genome had already been sequenced. Suppose the genome had been sequenced in a cross, and the whole genome of the fruit fly had been sequenced which it has been sequenced. And, we looked at a cross and we looked at the mutants. And what we did was we tried different positions along the genome. And at each position, we had some genetic marker./nAnd that genetic marker might be as simple as the fact that at that position, maybe there is an A in the DNA sequence on one of the chromosomes, and maybe I don't know a G in the other sequence. And over here, this marker might be, there's a T in some particular position, and there's a C in some particular position. If we could assay that, if we could tell, we could look whether this spelling variation is closely correlated with the mutant./nAnd this spelling variation is closely correlated with the inheritance of the mutant allele. And we could just try up and down the genome, different sites of spelling difference as if they were genetic markers in our cross because they are genetic markers in our cross, and see which one is most tightly correlated. The minute we get any genetic sequence difference, that shows co-inheritance linkage in this cross, we know that this spot in the genome must be nearby our mutation./nSo, we'll try one closer, and we'll try one on the other side. And, what you do is you test sites of genetic variation, first to find one that shows any co-inheritance. And once you've got that, you try ones closer, and closer, and closer. Last time I talked about the process of, if you had one of those markers you could use it to isolate the next clone and the next clone and the next clone. But you know what I realized? That's so old fashioned. We might as well deal with the fact we have a sequence of the genome./nNo more would you ever isolate the next clone and the next clone and the next clone. You just look it up in the computer. So, even if you have the whole sequence of the genome, we have to figure out what part of it was co-inherited along with this disease, and that's the way you do it, OK? Genetic mapping,just as Sturtevant invented it, can be applied if you have a whole sequence of the genome, and enough sites of variation. And, I've drawn it for a fruit fly cross, but this could equally well be cystic fibrosis. The only difference is if we're doing this in human families and it's cystic fibrosis we don't have as many offspring./nSo, we have to pool data from many families. And, we can't arrange it so that every family has exactly the same orange alleles up here and pink alleles down there, but computers can deal with that. They can still figure out the correlation across many families, and you find the spot in the genome where for many, many, many families the kids who all got the disease show correlated inheritance with this marker. And that eventually pins you down to a region of the genome. It pins you down to those genetic markers that show the absolute tightest correlation, tight correlation, and that's where you look./nAnd in that fashion, people went being able to map the location of Huntington's Disease in 1984 to, by now, mapping the locations of more than 1,000 different human genetic diseases where people didn't know the protein in advance. They did it entirely based on this positional mapping. So, Sturtevant's idea, which I like so much, has played itself out so beautifully now in the area of modern molecular medicine./nOK. So, onward. I want to talk about a few other variations on the theme rather quickly, and then I think I want to talk about how you analyze your clones. First, variations on cloning, I should just at least mention it. We talked about cloning in an autonomously replicating plasmid in a bacteria./nSo, you go to a bacteria. They have some autonomously replicated pieces of DNA. There are circles. You can clone in them, and you can typically, these things are on the order of, I don't know, 1,000 to 2,000 to 5,000 bases can be readily cloned in these plasmids. You can do more, but that's a typical kind of number is the insert size, typically. But we in the lab go up to much higher numbers like 10,000 sometimes./nYou can also, if you wanted to study yeast, it turns out yeast happily have plasmids as well, and you can do a similar sort of thing for yeast. It turns out that instead of using plasmids, you can use bacterial viruses. These bacterial viruses have all different shapes as we've talked about, circular or linear, and they can typically hold, oh, 15,000-40,000./nSome of these viruses are quite big. The bacteriophage lambda tends to carry a lot of stuff. And, it can replicate. So, you could do the same thing to that. You can even use viruses that infect mammalian cells and there are all sorts of viruses now that people clone in again, linear or circular. I don't know, for mammalian cells, you often, the viruses like 1,000-5,000. You can even make artificial whole chromosomes now./nYou can do this in yeast. Artificial chromosomes are called YACs. They have all the little machinery, little telomeres on them, little centromeres. They have a selectable marker, and then you can clone into it your piece of DNA. And these can take up to a million bases of DNA. So, if you wanted, there are bacterial artificial chromosomes./nThey're called BACs if they're in bacteria. And recently, people have developed artificial chromosome systems for mammalian cells, and specifically human cells. And they're called unfortunately MACs and HACs and things like that. Basically, any molecule that can replicate in any system, some smart molecular biologist will come along and say, how do I use that for my purpose, to stick my DNA in it, and get it to replicate in this organism?/nAnd so, if something's not on this list, it will be soon, OK? Now, here's another thing. This is cloning chunks of DNA. Just to have the piece of DNA in a library, but suppose we want to do more than just have the DNA sitting there in the bacterium, suppose what I'd really like to do is take a bacterium, E coli, and put it to work for us./nMaybe what I'd like to do is take a plasmid and insert in that plasmid the gene for human insulin. So, I'm going to take the DNA locus corresponding to human insulin, clone it into my plasmid. Maybe I'll have isolated it from my library because, let's see, insulin's protein sequence is known so I could reverse translate it to a nucleotide sequence./nSo, I could probe a library. So, I could find the clone that has insulin. Now what I'd like to do is persuade this bacteria not just to carry the DNA but to make insulin for me. Would that be useful? Yeah, how did people used to get insulin? Cadavers, dead bodies; it would be much easier to get them from a fermenter, right, to get insulin from a fermenter, if you could just ask E coli to make it. So, if we put it into E coli, will it make insulin for us?/nHere's the human locus, DNA for insulin. Will it make insulin? Let's see, how do you make a protein? You've got to start by making RNA, right? You've got to transcribe the gene. Will E coli transcribe this gene?/nWell, why? It's got a promoter, right? It's got the insulin promoter. There we go. The insulin promoter is here. So, E coli will come along to the insulin promoter and start making RNA? No, it turns out that promoters in humans and promoters in bacteria are sufficiently different. They don't work across species. They won't recognize the human promoter. Too bad. Any ideas?/nYep? Stick a bacterial promoter there. Good, you're acting like a good molecular biology designer here. Let's put a bacterial promoter here. It will recognize its own promoter. That's great. Then, let's put the DNA for the human insulin gene here. And now, maybe we'll put the Lac operon, and when it has lactose it'll start making RNA from the human insulin gene. And it'll start translating it./nAnd, we get insulin. Any problems? Well, will it make any, for starters? What's another aspect of mammalian genes that's different from bacterial genes? Processing, what kind of processing with the RNA? And the splicing, ooh, the insulin gene has introns that have to be spliced out./nSo, this is going to make some RNA, insulin RNA, and it needs to be processed like this. Will bacteria carry on our splicing for us? They don't do splicing. Yep? Well, that's a very interesting question because we haven't. But, what do you propose? You see, I've just taken a piece of human DNA from the human genome, which encodes the introns and the exons. But, you seem to have a solution to our problem, and what would that be?/nSo, instead of making a library of genomic DNA, what you're suggesting is a radical idea. Let's instead take human RNA. Here's some human RNA, lots of human RNA, a big collection of human RNA. What was at the end of the human RNA: a poly(A) tail./nAnd what I understand you to be suggesting is if we take human mRNAs, a whole collection of them, you want me to turn these mRNAs back into DNA and clone them instead of using the chromosomal DNA. How do I turn an RNA back to DNA? Is that possible? What do you use: reverse transcriptase. We have to give it a primer. So remember, five prime to three prime, we'd like to put a primer going over here. Any ideas for a good primer? Poly(T), isn't that convenient?/nOne of the reasons that mammalian messages have poly(A) tails is so that we are able to reverse transcribe them using poly(T) primers. No, that's actually not true. So, we use reverse transcriptase. And what we can do is we'll copy this RNA into a strand of DNA. There we go./nThen what we'll do, next step, is we'll take the DNA, and we'll copy back into a second strand of DNA. And now, we have double-stranded DNA whose sequence matches the already-processed mRNAs. Sorry? So, the sequences would match the mRNAs. So what you could do is instead of taking human DNA from the nucleus,/nyou could take RNAs, turn them back into DNA by reverse transcriptase, and make a library now that consists of zillions of inserts, each of which has what's called a cDNA, a copied DNA, copied back from the RNA. The great advantage of this is that the human cell has already done the splicing, and so there are no introns left./nNow, when you stick it in a bacterium, the bacterium is able to express this. It's able, if you give it its own bacterial promoter, to make an RNA. And if you don't ask the bacteria to have to splice, if you just give it a pre-spliced piece of DNA that doesn't need splicing, it can translate that DNA. Now, notice we used all of our tricks. You had to know about reverse transcriptase, poly(A) tails, structures of genes, introns, exons, yes, question?/nIt doesn't. You do this in the test tube. You purify human mRNA in the test tube. You take that mRNA in a test tube, add reverse transcriptase, add poly(T), make this reaction of RNA to DNA in the test tube go back. Where does it come from? Viruses that copy themselves back for a living, right? So, again, every single thing we're using comes from some living organism that does this kind of stuff./nAnd, when I teach you about the facts of how viruses replicate or what the structure of mRNAs look like or whatever, it's because every bit of knowledge we get about the way biology works turns into an incredibly powerful tool as it's turning out for us to actually be able to further study biology. So, great. So, where does reverse transcriptase come from now? Originally they come from viruses that turn themselves back from RNA to DNA. Now, how do you get reverse transcriptase?/nCatalog, right, very good. All right, so this is called, finally, a cDNA library. And, if you had made a cDNA library, you would be able to screen the cDNA library to find the gene for insulin. Is this useful? This happens to be, for example, one of the consequences of this was the biotechnology industry./nOK, so if you have any doubts about the usefulness of understanding these abstract things about E coli and bacteria and stuff like that, one of the consequences was Genentech, Biogen, and Amgen, and if you just simply walk around Kendall Square, within a mile of this place you will see laid out before you the consequences of this ability, OK? It's transforming Cambridge. Yes?/nAnd the world. Yeah. Indeed. It might be that producing large amounts of insulin was bad for the bacteria because there would be so much protein it would clump and kill the bacteria. It might be that insulin, for various reasons, might not fold appropriately in the bacterial environment./nAnd, this is why the biotechnology industry has lots of smart people working in it because you're totally, 100% right. You might decide that instead of cloning it in bacteria it's better to clone it in some insect cell in culture which, in fact, people like to work with, or some other cell, or a mammalian cell. And so, I simplify by saying put it in coli, but in fact that might test six different cell lines, six different host possibilities. They might have to take the insulin out and refold it in vitro and things like that./nYou're totally right. This is actually something that requires work to do it right, just like building an airplane requires work. I could tell you Bernoulli's principles, but then Boeing does more than just writes down Bernoulli's principles. OK, so onward. Now, I'd like to turn next to analyzing your clone. Analyzing the clone, so suppose we have, maybe it's by positional cloning, maybe it's by cDNA cloning, but one way or the other we've got us a clone that we're very interested in./nMaybe it has the insulin gene. Maybe it has the Huntington's disease gene. Whatever it is, we're going to want to study it. And at the moment, I haven't told you how I would even read its DNA sequence or analyze its DNA. So, the first step is, of course, I have to purify the plasmid. And, it turns out that that can be done./nThere are simple biochemical techniques, as I mentioned in a previous lecture, that allow you to grow up a lot of the bacteria, crack them open, and the plasmid being a little circle, and being a little more tightly super-coiled and wound up has somewhat different physical properties. And you can use those to purify the plasmid. So, plasmid preps are not hard to do. You can get a fairly pure collection of the plasmid. Now, suppose I've done this for, oh, I don't know, let's take my first example, orange mutants./nSuppose I tried to rescue bacteria that were orange minus, and suppose I found that 50 different plasmids rescued my orange mutant because I transformed a lot of plasmids in, I plated it, and 50 colonies grew up. Are they all the same thing or are they different? Is there any quickie way to take a look at these 50 plasmids and see if they're identical or fairly close, or obviously different? Well, I'd like to take some way to take the DNA from the plasmid and analyze it kind of easily. I might want to see, like, how big is the insert?/nRight, that'd be one way, if they had different sized inserts so they couldn't be the same thing. So, maybe what I could do is how do I clone this? I used EcoRI sites I recall. So, I have EcoRI sites here. Suppose I were to take this DNA, and I were to now cut the DNA from the plasmid with EcoRI./nThen, what I would get is two separate molecules. I would get the vector and the insert. How could I see how big they were? Gels, gel electrophoresis is the way to do that. So, I take a gel./nA gel is a slab of gelatin, Jell-O, OK, and normally it's laid flat, but I'm going to do it vertically here. I load into the top of it here a little bit of my DNA, this whole mixture. I take the plasmid. I cut it. I put it in here. DNA's positive charge or negative charge? Negative. So, where should I put the positive pull? On the bottom, well done. That's often not done, and to the detriment of the experiment. If you put the positive pull here, it goes the wrong way, and everybody has to do that at least once./nSo, what'll happen is the DNA fragments move through, and the smaller fragments move faster than the big fragments, right? If something's little, it'll move fast. If something's big, it moves slowly: little, big. Smaller moves faster because it wiggles through the little pores in the gel better. So, suppose I were to do this for a bunch of plasmids, and what I saw was this./nFirst order, what do you guess? Sorry? Top road's probably the plasmid vector. This is probably the vector, and what do I know about the inserts? At least two inserts, at least two distinct inserts./nNow, if I wanted to be sure that was the vector, maybe what I could do is take another row, and run a known amount of the vector, take the vector alone and I could check that the vector alone runs over here. And maybe I might take some other known molecules. These would be called molecular weight standards. So, if I run some knowns in one of the lanes of the gel, I can even measure and say, ah-ha, the insert is somewhere between the size of this one and the size of that one./nAnd so, I get a little ruler that I can put on the gel. So, in fact, that's the first thing you would do is you digest your clone that way. Now, does the fact that these guys have exactly the same, apparently, size on the gel mean that they're the exact same piece of DNA? No, because you can't even actually tell it's exactly the same. There's a limit to how precisely you can measure it./nSo, what else could you do? You could try another restriction enzyme. It turns out that since there are so many restriction enzymes in the catalog, if I take a piece of DNA, maybe that Eco fragment, I could try cutting it with HinDIII. And when I cut it with HinDIII, I'm going to get three distinct lengths. I could try cutting it with, oh, I don't know, pick another enzyme, BamHI./nWhen I cut it with BamHI, I'll get some other lengths. And, how to get these lengths by adding these, by running them out on a gel and looking at their sizes. What if I added both HinDIII and BamHI to my test tube? I'd cut at both sites. So, I'd cut here, here, here, here, here./nSo, this is cut with HinDIII, here cut with BamHI, here cut with both and I could measure these lengths. So, suppose I gave you this as a computer problem, I have a string and it's an unknown string, and I cut it at two places and I get these lengths, X1, X2, X3./nAnd then I take that same string and I cut it at other positions, Y1, Y2, and Y3 are the lengths that result. And then suppose I now cut it at both of the sites, and I measure it, and I get Z1, Z2, Z3, Z4, Z5. If I gave you all those numbers, could you figure out where the sites must be? Probably. It turns out to be a reasonably doable computer problem, although it can get a little hard in places./nAnd you could try a third enzyme and a fourth enzyme, and it's a cute exercise to write yourself a little piece of code that will figure out where the sites are based on the lengths. The reason it occasionally gets funny what if Z3 and Z4 are exactly the same length and they run on top of each other in the gel, and there are special cases. But you can kind of reconstruct where those restriction sites must be just by writing a good piece of code that'll put these pieces together. This is called restriction mapping, and it's great fun. Everybody likes to do this once./nBut, it's only a limited amount of information, right, because you get where the sites are, and I guess if I gave you ten clones and they all had exactly the same restriction maps, the exact same positions of these restriction sites, you'd feel pretty confident they were the same clone. But you still wouldn't really know much about the clone other than it had two HinDIII sites and two BamHI sites, and here's where they were. What do you really want to know about this clone? It's DNA sequence, right?/nLet's not settle for anything less than the exact nucleotide sequence of the clone. So, that's really the last key topic is sequencing DNA. How are you going to sequence DNA? Well, suppose I give you some double strand of DNA, five prime to three prime, five prime, three prime, double stranded DNA./nLet me heat it up. What happens when I heat up DNA? It melts the hydrogen bonds, the non-covalent hydrogen bonds here break, and I got my two strands separated. Now, what I'd like to do is I want to start reading out this DNA sequence./nSo, I'm going to make me a primer. Now, golly, here's a primer. You're going to ask me, how did I even know what primer to use if I don't know the DNA sequence? How can I make a primer? Hold that question. Make sure I remember to come back and answer that, OK? But for the moment, grant me that I have a primer here./nWhat I'd like to do is add DNA polymerase. So, let's add some DNA polymerase. And, I'd like to add nucleotide triphosphates, dNTPs, the dATP, dCTP, the dGTP, dTTP, and if I add DNA polymerase and I add my nucleotides, what does Arthur Kornberg tell us will happen?/nIt'll start polymerizing, right? And, it'll stop there. So, the polymerase knows the bases, right? It knows what base to put in because polymerase is very smart. So, the bases get put in correctly. The only problem is, how do we get polymerase to tell us what it just did?/nHere's a cute trick. This is, by the way, a cute trick that won the Nobel Prize. So, suppose my primer is like this: five prime, T, A, A, T, T, C, T, and the template strand here, A, T, T, A, A, G, A, now let's keep going, A, T, G, C, C, A, A, T, G, G, A, T, T, A, five prime./nSo, there's my primer. There's my template. I'm going to start adding. Well, let's add our polymerase./nLet's add our dNTPs, polymerase, dATP, dCTP, dTTP, dGTP, and then I want to add a special extra good old ingredient into this./nThe special extra ingredient I want to add is a defective T, a defective dTTP. What do I mean by defective? I mean chemically modified in such a way that it can't be extended, that you can't extend past it./nSo now, let's follow my reaction. I'm going to start with, I'm just going to write them down here, T, A, A, T, T, C, T. What's the next base I'm going to put in? T, OK? Is that a defective T or a good T? I don't know./nIt could be. Maybe it's a defective T, which I'll put a little star there, OK? If so, what happens to my polymerase? It stops. It can't go any further. It can't go any further because the T's defective. But what if it wasn't a defective T? What if it was a good T? Then what goes on? The polymerase will put in, keep going guys. A, C, G, G, and what does it put in now?/nT, right? Now, is that a defective T? Maybe. We don't know. If it is a defective T, it stops there. Otherwise, polymerase goes here, and the next space is what? T, and is that a defective T? Maybe./nAnd, if it's not a defective T, then polymerase goes on, puts in an A, puts in a G, a C, C, and then a T. And maybe that's defective. All right, which of these possibilities is what polymerase does when I throw it in?/nWell, all of them. There's a lot of molecules there. Some of the molecules, by chance, happen to install a defective T, and they grind to a halt here. Sometimes, a good T's put in and the molecules stop here. Sometimes they stop here, and if I start with a big collection of primers in a lot of my template DNA, I'm going to get this whole collection of different molecules of different lengths. What lengths do I get? The lengths correspond precisely to the positions of the Ts./nI get a series of molecules whose lengths perfectly match the positions of Ts. Well, first off, how do I measure their lengths? Run a gel, bingo, run a gel./nSo, if I could run a gel that could separate nucleotides based on length that two next to each other, another one up there, I'd see a small molecule, length one, two, three, four, five, three, six, eight, I'd see one of length eight. I'd see one of length, what's the next one, 13, eight, nine, ten, 13, 14, so eight, nine, ten, 11, 12, 13, 14, 15, what's that, 13, 14, 15, 16, 17, 18./nOK, those would be the positions at which I would see this T. So, I'd need to have a special kind of gel that's so accurate that it can separate single nucleotides, right, that the lengths, but that can be done./nThere's acrylamide, the polymer that will do that. That'll tell me the exact lengths of the T's. What else do I do? Well, let's obviously do it from the other bases. Let's try defective A, defective C, defective G. Let's see, if I got it right, which we'll try, it ought to end up looking something like that./nAnd if not, you get the picture, that this ought to match up as to which columns have which lengths. OK, I think I got it right. That tells me the lengths of the molecules. So, I could read off at sequence. The sequence of that molecule ought to be, starting over there, the sequence of what I've added in, ought to be something like T, A, C, G, G, T, T, A, C, C, T, yep, it worked. It's exactly right. Bingo./nI can now read the sequence. Fred Sanger, a brilliant scientist, thought up this method of just exploiting E coli's own polymerase or other organism's own polymerases. So, copying and all the chemistry that had to be done was thinking up a defective nucleotide that could not be extended./nIt could obviously be inserted. It can't be extended. So, one question is, what's a defective nucleotide? Well, you will recall that our nucleotide in the sugar phosphate chain is sitting like this./nLet's see, hanging off the one prime carbon is the base. This is the one prime carbon, the two prime carbon, the three prime carbon, the four prime carbon, the five prime carbon. What do we know in DNA at the two prime carbon? Normally in ribose there would be a hydroxyl here, right? But in deoxyribose, there's just a hydrogen./nSo, if this is deoxyribose, so a dNTP really means a two prime deoxyribose, where do I now attach my next base in the sugar phosphate train? Three prime ends, and what do I attach it to: the OH./nWhat do you think would happen if there's no OH there? You're stuck. All you've got to do is take off that hydroxyl. No hydroxyl group. If you made nucleotides that don't have that hydroxyl group, they can't be extended. So, instead of these being just deoxy at the two prime position, they are dideoxy, deoxy at two positions./nThey are two prime, three prime, dideoxynucleotides. That's it. Now, if you needed to get two prime three prime dideoxynucleotides, they're in the catalogue of course, right, because Fred Sanger had to make them himself and all that, but you can just buy them now. And so, you can do the sequence. A few other little details here, though, guys./nHow do we see the DNA and the gel? One possibility would be staining it. There are some dies like ethidium bromide, and for doing your restriction mapping, using a dye that sticks to DNA like ethidium bromide does is pretty good. And then you put it under fluorescent light and you look. For sequencing, the amount of DNA is so little that it's hard to see with a dye by the naked eye, which is what you do with restriction map. So, sorry?/nSo, the first thing people did was radioactive. What they did was they took a primer, made it radioactive, and you did this whole sequencing reaction with radioactive primer. Then, when you run the gel, you take your gel and you expose it for some number of hours, eight hours maybe, a piece of x-ray film, develop the x-ray film, and you'll see that picture. So, one solution that you could do to visualize is using radioactive nucleotides./nSo, we got the defective nucleotide. We now need to visualize our DNA. Let's visualize the sequence. One possibility: radioactive. The second possibility, someone already mentioned it, a fluorescent dye./nNow, here, a fluorescent dye could be put on, and you can't read it with your eye, but lasers are very good at reading. So, you might run a whole gel here and have lasers scan it. But, you can actually do better than that. Suppose I put my fluorescent dye on my dideoxynucleotides./nSuppose I put it on my dideoxynucleotides, and suppose I even had enough chemistry at my disposal that I could put a different color of fluorescent dye on each of my nucleotides./nThen, whenever the dideoxy is put in to terminate the chain, it carries with it its own color. Wouldn't that be cool? And, that's what's done. Not just can you buy dideoxynucleotides now, but you can buy the four different dideoxynucleotides each with its own dye attached to it. So, there are di-dideoxies I guess, sorry, but it's different di's, right? They're dye-dideoxies. So, you could do that./nAnd then what you get would be that in this column you get this color. And in this column, you'd get this color. And in this column you'd get this color, etc.? I'm not worrying about where they are here. And they'd all be different colors and it would be very pretty. You know what? Why do we need to run separate lanes anymore? If we got a laser, we can tell the laser scan it to tell it different. Stick it in one way./nIn fact, what's done is stick it in a capillary tube, throw in all four at the same time now, and as these fragments come by, each has its own color. And all we need is a laser scanner capable of sitting right here. Here's my laser scanner./nAnd the laser scanner, positive here, negative here, as the DNA flows by through this polymer, the laser scanner reads off which colors just went by. And it goes A color, C color, T color, G color. That's it. So, there are actually machines now that have 96 different capillaries. These are called capillary tubes./nAnd you can have 96 of them with laser scanning across, and in each column now, it turns out that you can read almost 1,000 letters, 1,000 bases per column per capillary times about 100 capillaries. Or in other words, you can read out about 10^5 bases of information./nYou can read out 10^5 bases of information in about two hours. Of course, you can do that ten times a day. So, you can actually read out 10^6 or about a million bases of information per machine. And here at MIT, we have 100 of these machines. So, we actually can read out a little shy of 100 million letters of DNA sequence per day, which I mean is a lot./nWe read about 40 billion letters per year here at MIT, and this is how we do it. How much does a machine cost? List, or do you want a deal? They list for $300,000, but if you buy in bulk, I can do better. [LAUGHTER] We buy it in bulk, by the way./nSo now, how are we going to get our primer there? That was the only little bit we were missing is where did our primer come from? The last little detail: here's my vector, remember, and I want to sequence this insert. How am I going to get a primer in the insert? I don't know what its sequence is. How do I even start this? Sorry? Well, but that won't tell me what the sequence is that I have to, I mean, I was looking to try to get a primer that matches the insert./nAnd I don't know what the insert is. So, how am I going to get a primer? Oh, I know the vector. The vector is well known. It sequence is in the catalog. Let me instead just use a primer that happens to sit in the vector, and I'll match to a known sequence to start with, and then I'll sequence into my unknown territory. So, this is how you get the initial primer was you arrange that your initial primer is sitting in known vector sequence./nAll right, so you can now sequence DNA. I've got to say, I've taught this course for a little more than a decade, and being able to say, now we can routinely sequence about a million letters per machine, and 100 million letters per day, and things like this was not routinely the case. When we started teaching this course, I was describing what we did, and it was the radioactive sequencing, and it was really quite impressive if a lab could manage to get a couple hundred letters a day./nSo, progress does happen within real time. This is what sets this apart, say, from introductory calculus or something like that. Good bye, guys. Everybody think good thoughts about the game tonight. www.mit.edu
Tags // Recombinant DNA Lecture
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Recombinant DNA Lecture 1
Biology > Introduction to Biology/nVideo Lectures - Lecture 15/nTopics covered: /nRecombinant DNA 1/nInstructor: /nProf. Eric Lander/nTranscript - Lecture 15/nGood morning. So, I see we have a lot of parents here. How many parents have we got here? Welcome to the parents. How many of the parents have done the reading for today? [LAUGHTER] Good, because we'll call on the parents too, right? We'll see what happens./nAll right, so, where are we? We've talked about this diagram that I keep coming back to. If you want to study biological function the two traditional ways to do that were to look at genetics or to look at biochemistry: genetics, the study of an organism with one broken component, those components being genes; biochemistry: the study of the purification of individual components from an organism away from the organism, particularly the most important such components being proteins./nWhat do they have to do to each other? The unification in molecular biology that occurred in the middle of the century from the 1950s into the ‘60s and really up to 1970 or so, we came to a conceptual understanding that genes encode proteins, and therefore these two different ways of looking at the organism: organism minus a component, components minus an organism were complementary points of view, and in theory, you could go from a gene sequence to a protein sequence, a protein sequence back to a gene sequence, to go from a gene sequence to its function, its function to a protein, except for one to a tiny detail./nThis was all just conceptual. Conceptually we understood by about 1970 that the DNA made the RNA made the protein. The protein carried out the function but as of then, you couldn't individually work with or purify the DNA corresponding to any particular gene./nAll of the inferences had been indirect inferences: indirect inferences from bacterial genetics, bacterial regulation or Meselson-Stahl experiments, and all sorts of interesting indirect ways working out the genetic code, but it didn't let you read anything./nThis was a problem. Some people in the late 1960s said, great, molecular biology is over. We understand in principle how life works. Now let's go understand how the brain works. And there was an exodus of some people from molecular biology into neurobiology to now go nail the brain, figured that would be worth another ten years or so./nBut in fact, remarkably, people began to focus on how you could get to work with individual specific genes. Now, what's so hard about that? I mean, it's not very hard to crack open a red blood cell and purify different proteins./nYou can purify hemoglobin. You can purify different enzymes. Biochemistry allows you to purify different components from each other. I want to purify an enzyme: let's crack open a yeast cell, separate the proteins over some column that separates them based on their size or their charge, and I'll get purer and purer fractions./nI'll assay each fraction to see which one has the enzymatic activity. But basically I use the physical chemical properties of the proteins to separate them into different buckets. Why not do that with, say, the human DNA and purify out the gene for beta-globin, that encodes the beta component of hemoglobin? What would be the problem of just using physical chemical purification to purify one human gene from another? Well, I mean, it's one very big molecule./nWell, I could shear it up. Maybe I'll just break it up. Now, let's purify the beta globin-containing part. It all looks the same. It's just DNA. It's one chemical polymer with pretty boring properties, and they're not very different./nAny particular DNA sequence in any other DNA sequence basically about the same molecular weight, same charges, there's nothing to separate them by. How are you going to purify beta-globin? That was the problem./nThat's where recombinant DNA came in was recombinant DNA was a remarkable and totally different way of purifying individual components. And the basis of it was this notion of cloning. If I want to purify out from the human genome, how big is the human genome? The human genome is about three billion bases long./nIf I want to purify a particular gene, let's say beta-globin or some other gene, typical gene, might be on the order of 30,000 letters long. This is one part in 10^5 purification I've got to achieve./nAny given gene is only one part in 10^5 of the human genome. And then, what about a typical mutation? Maybe the mutation that causes sickle cell anemia by changing a single nucleotide in beta-globin, well, that's one base pair./nSo that means I'm trying to identify something that's on the order of one part in 10^9 actually, a little less than one part in 10^9 of the whole genome. Carrying out purifications like that is really kind of hard to imagine./nBut the way it was done was by the invention of cloning. Let me briefly overview of the idea of cloning, and then we'll dive into the details. The idea of cloning was, the way to purify individual molecules would just be to take the molecules and just dilute them so that there was only one of each model./nThat's very pure, isn't it? The problem is it's not very much, so you need a way to take a single copy of a molecule, and then make many copies of it. So purification's not hard. You just dilute it down so you work with single molecules but then you need to copy it back again and again and again, and no biochemical technique involves, say, fractionating a cell and replicating some enzyme, you know, copying some enzyme./nYou can't copy enzymes, but you can copy DNA, and that was the basis of it. So here's the way it goes. The basic overview we'll look at is take your DNA and cut your DNA of interest, maybe the human genome, into pieces at defined sites Then, paste your DNA, which is more technically ligate, the word we use./nPaste your DNA to some other DNA called a vector. So, cut your DNA and paste your DNA. Each piece of your, say, human DNA gets stuck to some piece of vector. Insert this DNA into vectors that can replicate in bacteria./nSo, I'm going to actually take my piece of human DNA and not just ligate it to any piece of DNA. I'm going to take my human DNA, and I'm going to ligate it to a vector that has all of the machinery, all of the ability to be copied in a bacteria./nThen what I'm going to do is I'm going to transform my DNA into a host cell, a host bacterial cell. Transform means introduce. When we talk about transforming DNA, we're not talking about changing it./nIt's the word that's used for taking my DNA, stuck into a vector, and introducing it into bacterial cells. Ideally, each bacterial cell would carry one such DNA molecule, and then what I want to do is I want to plate my cells, and select those that carry human DNA, my DNA; DNA I've put on it./nSo, I'm going to put them on a Petri plate and I want only the bacteria that happen to have picked an individual piece of human DNA to grow. So, that's the trick. It's a very simple trick. Take total human DNA, cut it up into pieces, glue it to a vector that's able to be copied so that it's able to be replicated in bacteria, put the vectors into bacterial cells; every bacterial cell picks up no more than one vector./nYou plate it out, and you simply arrange so that the only cells that grow are those that picked up the piece of human DNA. And then, every one of these colonies is the descendent of a single bacterial cell that picked up a single human molecule, but is obligingly copying that molecule for you again and again and again and again./nAnd thus, you have what we refer to; this whole collection here is called a library of clones. This is called a recombinant library because every piece of the human genome is somewhere in here. You know, this one here probably is actin, and maybe this one here maybe is collagen-11 and that one there might, ah, there's beta-globin./nOK, actually when you look at the plate there's no way to tell but in principle they're all there. So, there will be this question of, how do we look at a library and pull out what the right one is? But somewhere in there should be a bacterial colony that has pure beta-globin gene, the DNA for beta-globin./nThe next lecture will be about how you actually find it. But today let's just build this library. So our goal is to be able to build a library like this. So, we have to figure out how to cut DNA, paste DNA, vectors, etc., etc./nSo that's what our subject will be today. Let's dive in. First, cutting DNA, how do you cut DNA? Restriction enzymes, etc. It turns out that the way you could cut DNA at particular places is as follows./nLet me take a piece of DNA. Here's a double-stranded piece of DNA. We'll go A, G, C, T, A, G, A, A, T, T, C, T, T, A, C, C, hydroxyl there, three primed. Let's go back on the other strand. What do we have? G, G, T, A, A, G, A, A, T, T, C, T, A, G, C, T, hydroxyl there, three prime./nThere's my double stranded piece of DNA. It turns out that there exists an enzyme that recognizes that exact sequence: G, A, A, T, T,/nC. The enzyme goes by the name EcoRI. This protein, this enzyme, scans along the the DNA, and it finds this sequence: G, A, A, T, T, C./nActually it's on this strand. What about on the other strand does it say? Same thing. But it's a reverse palindrome. It's symmetric. That's very good. And it turns out most restriction enzymes do that./nOK, so what it does when it finds that, with the benefit of colored chalk that has just shown up here is it cleaves the DNA fragment like that. And what it gives you then is a broken double strand with an overhang, T, T, A, A, five prime, three prime, three prime, five prime./nThis has a hydroxyl here this. This has a phosphate there. And then this other fragment here is A, A, T, T, C, T, T, A, C, C, G, G, T, A. So, what happens is, and this has a five prime, three prime, three prime, five prime I get two fragments of DNA that have been broken there and have it over./nThe overhang is complementary. Those two sequences match each other. There's what's called a five prime overhang and they're complementary So, we have complementary, that is matching, five prime overhangs./nThis is called EcoRI because it's purified, this particular enzyme, from E coli strain R and it's the number one such enzyme that was purified from it. So, it is very simple nomenclature here. Now, here's a question./nWhy do bacteria have an enzyme like this? There are some people who feel that the reason is that this enzyme is here precisely to allow molecular biologists to cut and paste DNA, and this represents impressions likely, me among them./nHow did anybody find this stuff? Well, shaggy dog story, I have to tell you the following shaggy dog story. So, this is a fun shaggy dog story, and it's an MIT shaggy dog story because it comes from the work of/nSalvador Luria, who is a very famous biologist who worked here at MIT./nSo, Salvador Luria was studying bacteriophage. Remember, bacteriophage are the viruses that infect bacteria. So, he was studying bacteriophage, and he took his bacteriophage and used it to infect a strain of bacteria, strain A, and he also used it to infect a strain of bacteria, strain B./nSo when he did that, what you do is you plate a lawn of bacterial cells. You kind of have a slush of bacterial cells that you plate here with virus mixed in, and wherever there's a virus, the virus grows, replicates, and either kills or slows down the growth of the cells so that bacterial cells grow everywhere else, but where a viral particle landed there's an absence of bacterial cells and that hole in the lawn, this whole thing is called a lawn of bacteria, and the holes in the lawn are called plaques./nSo, when he did this, he found that when he did it on strain A he got a bunch of plaques and when he did it on strain B, he didn't, no plaques. So what what's the simplest explanation for this? Strain B is different somehow./nIt's resistant to the virus. I don't know, the virus has to come in and do various things, and strain B isn't compatible with the virus or something like that. No big deal. So it's a resistant strain./nBut, occasionally you'd get a plaque. Very occasionally, you'd have an occasional plaque. So now, how would this be? I said the strain was resistant. How could there be an occasional plaque? Mutation in, could it be a mutation in the bacteria? Sorry./nWell, if it was a mutation in the bacteria there would be one bacteria that had the mutation. It was now susceptible, and it would die. But, the lawn wouldn't kind of grow because the cells around it wouldn't have a mutation./nSo it's probably not a mutation in the bacteria but what could be? Maybe a mutation of the virus: what if it was a mutation in the virus that was able to overcome the resistance? Ah, so that's OK. So, what this must be is the existence of a resistant virus that is a virus that can overcome the resistance of the bacteria./nSo far: perfectly normal, no problem. Now, let's do the followingexperiment. Let's take this resistant virus, and grow it, again, onstrain A and grow it on strain B. What do you think is going to happenwhen I grow it on strain A? It'll grow lots of plaques./nIt still grows on strain A, and now what's going to happen when I growit on strain B? If this was really a mutation that made it able to growon strain B then it gets lots of plaques because it's now gained theability to grow on strain B, and sure enough, that's what happens./nSo, there's nothing funky yet. But now, suppose I take one of theseresistant viruses that I isolated here on strain B, I grow it again hereon strain A. It grows. I grow it on strain B. It grows./nIf I take it again from strain B and I repeat this, it'll still grow on strainA and still grow on strain B. Let's take one, though, from strain A. It'sthe resistant one which we have just now happened to have grown onstrain A./nAnd now, let's grow it again on strain A versus on strain B. And sureenough, it continues to grow on strain A, no problem. And we grow itnow on strain B. And, what shall we get? Well, it should grow onstrain B, right, because it was a mutant virus, and it gained the abilityto grow on either./nWe passage it through B, it grows. We passage it through A. But theanswer was nothing, no growth. How can that be? We had a virus.We agreed that was a mutant virus that had picked up the ability togrow on strain B, and we demonstrated it has now on either A or B./nWe then reached in, and grabbed a copy of it here from strain A,having grown on strain A, and we try it again and it now won't grow onstrain B. If this was a mutation, I mean, maybe the mutationreverted, right? It was a reversion of the mutation./nIt mutated back. Is that plausible? No, come on. The chance that allof the copies there would mutate back, come on. I mean, you couldrepeat this several times and this is always what happens. What doesthat tell you about this mutation in the virus? It can't be a mutation ofthe virus because if it was a mutation, it would be transmittedthrough./nBut, passing through strain A makes it lose its ability to grow on strain/nB. But as long as you keep passing it through strain B, it can grow on/nstrain B. This is not your typical genetics. So, Salvador Luria loved this./nAnd, he really worked out what was going on. And somehow, well, so anyway, they referred to this as strain B having the ability to restrict the growth of the virus. Strain B can restrict the growth of the virus./nThat's where this word restriction enzyme comes from. What's really, truly going on here underneath the shaggy dog story? It took a long time before the shaggy dog story that Salvador Luria was the one to really demonstrate is fully worked out./nBut, what turns out to be the case is that strain B has a restriction enzyme. That's how it restricts the growth. It has one of these enzymes that can cut DNA at a specific place. When the virus comes into strain B, it injects its DNA, and the enzyme comes along and cuts the virus's DNA, protecting the bacteria./nIt's got its own little defense mechanism: pretty cool, pretty cool. So, any DNA that's introduced, if it has the sequence here, it'll take G, A, A, T, T, C, the bacteria cuts it. Wait a second, the bacteria has its own DNA./nWhy doesn't it chop up its own chromosome? Well, I mean, so one simple possibility would be that if this thing is looking for the sequence, G, A, A, T, T, C in the genome, maybe it's the case that the bacteria has arranged that its own DNA never has a G, A, A, T, T, C./nThat would be a simple solution, right? But is it a plausible solution? Why not? But just statistically, how often do I expect to encounter a G, A, A, T, T, C? What's the frequency of any given six letter word in a four letter alphabet? It's about one in 46./nSo, about one in 46 positions will be a G, A, A, T, T, C, and that's about 4,000 letters. So, every 4,000 letters, I expect to encounter a G, A, A, T, T, C. How big is the E coli genome? 4 million letters./nSo, how many G, A, A, T, T, Cs will there be? About 1,000 of them. It's just not plausible to imagine that it doesn't have the sites. So, your idea is that if it has these sites, it's got to arrange to protect its own sites./nSo, how is it going to protect its own sites? Covers it or something. You could imagine something covers it or something, but you want to/nalter your own, so it turns out you're exactly right. What happens is there is an enzyme that comes along, and at this position, attaches a methyl group./nIt modifies the DNA by attaching a methyl group. It turns out that that methyl group is enough to prevent the restriction enzyme from binding. So, this blocks the restriction enzyme. So, that way the bacteria is able to distinguish between its own DNA, which is methylated, and the viral DNA./nSo, wait a second, how does that explain my virus that manage to grow? How did my virus manage to grow? It would need to have gotten itself modified also to be protected. Could that happen by chance? What if the methylation enzyme, the methylase, which is floating around in the cell, "accidentally" methylated the virus's DNA? What would happen then? The virus would become immune./nSo, suppose the bacteria was pretty clever, and had a lot more restriction enzyme, and only a little bit of methylase? Well, you'd imagine that most of the time the restriction enzyme would cut up the viral DNA first./nBut every once in a while, the methylase would get there first and protect the virus's DNA. That becomes an immune virus because it can't be cut by the enzyme anymore. And, if I take that, and I grow it again on strain B, it'll now produce lots of plaques because it was methylated./nAnd, if I grow it again on strain B, it remains methylated because once it's methylated and comes into the cell, it's not cut. And so, its descendants will get methylated. But, what happens if I ever grow that methylated virus on strain A? Strain A doesn't have the restriction enzyme, and it doesn't have the methylase./nSo, the progeny phage that grew up on strain A aren't methylated. They're no longer protected. The protection that the virus has is the protection that comes from this methylation enzyme. It's not the sequence of the DNA./nIt's the attachment to these methyl groups. And so, it turns out that if you ever pass this virus through strain A, passage through strain A, the resulting DNA loses or is unmethylated. And now, it can be cut./nAnd it can be cut. Well, this explained the weird results of Luria, that somehow bacteria had a complex defense mechanism of a restriction enzyme and a cognate methylase. The restriction enzyme would cut the sequence./nThe chromosome would be protected by methylating that site, and usually it would work fine. Occasionally the bacterial virus would get methylated. It would be protected as long as it continues to go through strains that have this restricted methylation system./nThat was it. Now, this shaggy dog story took a couple of decades to work out, and eventually led to Nobel prizes for the discovery of restriction enzymes. They're extremely important because although bacteria do this to protect themselves, they have also given us the perfect tool to now cut DNA where we want to cut DNA./nNow, what if you wanted to cut at a G, A, A, T, T, C? You've got EcoRI. But what if you wanted to cut it cut it in another sequence? Well, it turns out that if you want to cut it at G, G, A, T, C, C there's an enzyme called BamHI./nIf you want to cut it at A, A, G,C, T,T or A, A, G, C, T,T, there's an enzyme called HinDIII. If you want to cut it at just G, A, T, C like this, C, T, A, G, an enzyme called MboI. And, there are enzymes that cut it this way, enzymes that cut it this way, enzymes that cut it this way, enzymes that recognize four bases, six bases./nThere are even enzymes that recognize eight bases. It turns out that bacteria have elaborated zillions of different restriction enzymes that recognize different sequences. This is perfect for molecular biologists./nBacteria, of course, are much smarter than we are, having been at this much longer, have developed all of these tools for engineering. All we have to do is borrow them. So how do you get EcoRI? We grow out that strain of E coli; you purify EcoRI./nAnd how do you get HinDIII? You grow up strain of haemophilus influenza. You purify the enzyme. At least, that's how primitive molecular biologists did it. If you wanted to work with a restriction enzyme, you'd grow up the bacteria./nYou'd purify the enzyme yourself, and you would just use it in your laboratory. Of course today what does a modern molecular biologist/ndo if he or she should want HinDIII? It's in the catalog. So the catalog has 200 restriction enzymes./nYup, PsiI is new, on sale, 500 units for $400. Let's see what EcoRI is going for. Eco R1: look at this, 50,000 units $200. That's a good price for EcoRI because it's a very famous enzyme here. So all you have to do is you give them your credit card number and you have it tomorrow by FedEx./nSo that's how restriction enzymes are obtained today. So, next up, we can cut DNA any place we want to. We now need to glue DNA together. Suppose I cut DNA, human DNA, and I'm going to cut it. I'll just take human DNA, your DNA, which I've purified, and I'm going to cut it at all its EcoRI sites./nI can take any other DNA I want. I don't know, I could take zebra DNA. I could take anything and I could also cut it at EcoRI sites. I could mix them together, and after mixing them together the fragments will float around and remember this down here has T, T, A,/nA./nThis fragment over here from some other piece T, T, A, A, this could be human DNA. This could be zebra DNA if you want to. It doesn't matter. It could be bacterial DNA. These fragments overlap. They'll hydrogen bond a little bit, but that of course won't introduce a covalent bond here./nI'd really like to make a covalent bond. I would like to attach the piece of DNA from one source to the piece of DNA from the other source by doing the opposite of the restriction enzyme. The restriction enzyme cut at these locations./nI would now like to catalyze the rejoining of the sugar phosphate backbone here. So I would like to rejoin the sugar phosphate backbone. I have a hydroxyl here. I have a phosphate here, and I would like to ligate them together./nSo how I manage to ligate? What kind of fancy chemistry do I do to ligate these pieces of DNA together? I don't do any fancy chemistry. I again sit at the feet of bacteria who have solved all these problems before./nAnd I ask bacteria, how do you do this? And they say, well, we have an enzyme called ligase. So, you purify ligase from bacteria, you add/nthat, and ligase ligates the fragments together. Why do bacteria have an enzyme ligase? For repair of their own DNA./nThings go wrong, this is part of the DNA maintenance scheme of bacteria. They have an enzyme ligase to repair their own breaks in DNA and, obligingly, you can purify DNA ligase. So you add ligase, today, of course, if you need a ligase, how do you get it? It's in the catalog, absolutely./nSo, you can glue together any of those things you want. All right, next up, what DNA do I want to stick together? I mean, here I made a silly example. I'm going to stick some human DNA to some zebra DNA./nWhy do that? I mean, just to show you that I can doing it, right? I'm just demonstrating that I could stick any DNA to any DNA. Remember, once I've got a piece of DNA it doesn't know whether it came from a human or a zebra./nIt's just the molecule. You can stick the molecules together, right? But what do I really want to attach my human DNA to? I want to attach it to attach it to some other DNA that has the ability to grow on its own within bacteria./nVectors: I need to make, here's what I would really like. I would like to have a piece of DNA that has some sequences that contain the recognition sites for replication. I'd like to have some replication initiation sites here./nSo, a piece of DNA that, remember, because the bacterial chromosome itself, here's my bacteria, the bacteria's own chromosome replicates itself, and it has the ability to start DNA replication at multiple sites called origins of replication./nBut, what I would really like is to be able to construct in the laboratory a synthetic piece of DNA that also would function as an origin of replication because then what I could do is in vitro take my piece of DNA, attach it to this vector, and it would now have the ability to grow the bacteria./nHow am I going to make a piece of DNA? What kind of engineering tricks can we do to create a small piece of DNA that has all the machinery needed to be able to be copied and replicated just like bacterial chromosomes? That's a pretty fancy feat of engineering./nHow are you going to do that? Sorry? OK, so who are you going to ask? If you wanted to do this, you're going to ask the experts. Who are the experts? Viruses or bacteria, or basically, if you want to do anything, the place to ask is the folks who have the most experience./nAnd, the folks who have the most experience are almost always prokaryotic organisms because they are by far the most evolved things on this planet. Anything that can replicate itself and grow every 20 minutes or something like that has had a lot more generations of evolution than you have./nAnd therefore, they are much more optimized than we are. And so you go ask and say, has any bacteria worked out how to do this? Turns out bacteria have worked out how to do this just fine. In fact, most bacteria, at least many bacteria, contain within them, in addition to their own chromosome, small circles of DNA./nThese are called episomes. This is the chromosome. Epi means on top of or in addition to. So in addition to the chromosome, there's an episome. The episome is in fact an autonomously replicating piece of DNA that has an origin./nAnd it replicates. Why do bacteria have episomes? It turns out episomes often contain genes. One of the genes they contain, or some of the types of genes they contain, are resistance genes. There might be, for example, a penicillin resistance gene contained on an episome, or a streptomycin resistance gene./nIt turns out the bacteria have these episomes containing resistance genes, and they're not in the chromosome. They're separate. Now, why would they do that? It turns out when a bacterium dies and a cell cracks open, the DNA spills out./nThe next door neighbor bacteria has mechanisms to suck up DNA from the environment. You never know. It might find something interesting out there. So, it turns out that bacteria are rather promiscuously exchanging pieces of DNA all the time./nAnd so, a bacteria that has an episome that has a penicillin resistance gene can spread it to other bacteria, and it's very nice. It's compact. It's on its own little episome, autonomously replicating piece of DNA./nThis is great for bacteria wanting to spread drug resistance. It's not good for human populations, for example, because this is how drug/nresistance spread through populations. This is why we have spreads of penicillin resistance./nNow, of course, wait a second, this whole mechanism of spreading drug resistance, we've only had antibiotics since the 1940s. How did bacteria devise this so quickly? Sorry? Many generations since 1945? That would be very impressive./nYeah, but, I mean, why do they have this episome mechanism, the ability to spread DNA and all that? That's an awful lot to evolve in 50 years? Yeah? Something natural like penicillin. It turns out, we didn't think of penicillin./nWho thought of penicillin? Fungi. Right, again, we learn from the lower organisms. Penicillin comes from fungi. Bacteria have been fighting off penicillin for millions and tens of millions of years./nSo, we may be very proud of our penicillin and all that. But, they've been at this for a very long time. This is about war between bacteria and fungi. That's what this is, OK? So, that's why these things are here./nThey're here so that bacteria can have these resistance genes against fungi and things like that that make antibiotics. Antibiotics are natural. We've made a few new ones, but most of the antibiotics have been made by nature./nAnd so, if I wanted to replicate DNA, if I wanted to attach my human DNA to a piece of DNA that's capable of autonomous replication, autonomously replicating circles of DNA, these autonomously replicating circles of DNA are also called plasmids./nAnd that's the word we'll mostly use for them, plasmids. All I need to do is purify a plasmid from a bacteria. So, I find a bacteria that has plasmids. I purify the plasmid, and then I can cut open the plasmid at the EcoRI site, OK? So, this plasmid will have an ORI, an origin of replication./nI'll cut it open at the EcoRI site. I'll take human DNA fragments that I've cut with EcoRI. I'll mix them with plasmid DNA that has been opened up, has an origin. Ligase will come along, join this up, and now I have a circle of DNA that has all the machinery to autonomously replicate, plus my human DNA./nNow, if I wanted to get a vector, or an honest to goodness plasmid, I can go to a bacteria, grow it up, purify the plasmid, and cut it. Or alternatively, if I needed the plasmid, say, tomorrow, it's in the catalog./nThe next section of the catalog has a long list of plasmids here. There's a plasmid there, right? It's a nice plasmid. Oh yes, let's see, pUC is a very good plasmid. pBR-322 is a good plasmid. The whole section, all this purple stuff are the plasmids./nSo, you can get the plasmids too. You place one order, you get the restriction enzymes, you get the ligases, you get the plasmids, no problem. So, I can then take total human DNA, cut up, cut up, cut up, cut up, add in plasmid, and I'm going to ligate together./nAnd then, having ligated my human DNA to my plasmids, I'm going to mix with bacteria. I take some bacterial cells. I add my mixture of these plasmids containing human DNA. And now all I have to do is persuade the bacteria to suck up my plasmids containing human DNA./nHow do I teach bacteria to suck up DNA? They do that for a living. That's what they do. They're always spreading material. They have that ability. All we're doing is we're using their ability. So you get the sense that the kind of engineering that really works in biology is engineering that exploits what nature has been doing for a very long time./nRather than butting your head up against the problem, usually somebody has solved it, and it's almost always bacteria. So, you've transformed the bacteria. Now, there are a few tricks you can use to make them a little more transformable./nYou can add calcium phosphate, and blah, blah, blah, but you can sort of persuade them to take up the DNA. And then all you have to do is plate them out on a plate. Plate them out fairly dilutely so there are a lot of single bacterial cells that land on the plate, and wait for them to grow up./nEach one of these had a single plasmid, a different plasmid than the next guy over. Wait a second, each one? How do I guarantee that every bacteria in my test tube took up a plasmid? Is that plausible? I mean, I can't guarantee that every bacteria is going to take up a plasmid./nMaybe I'll add so much plasmid that every bacteria will take one up. Oh, but that's a bad idea because why? Because then a lot of them will take up more than one. You don't want to do that. You really only want to have at most one./nSo, if you were going to arrange so that at random you only have about one, you've got to have a lot that are zero. So, this is a problem. I mean, it's a real waste. My library is going to have large numbers of bacteria that don't have any plasmid./nIn fact, this transformation process is not so efficient. It's not so efficient. So, we have a little bit of a problem here is that some of these guys will have human DNA. But, most of them won't./nSo, what can I do to arrange that any bacteria that did not pick up a plasmid was incapable of growing? Add a resistance gene to the plasmid. Suppose I were so clever as to add to that plasmid, penicillin resistance./nSo, not just an origin of replication, but suppose I also had a resistance gene here, say, for penicillin resistance or streptomycin resistance, or ampicillin tends to be a very big favorite, ampicillin resistance./nThen, my plasmid would have ampicillin resistance gene encoded on it, an enzyme that can, say, break down ampicillin. So, what do I do to my Petri plate? I just add ampicillin. Now, even though most of the bacteria have not picked up a plasmid, only those bacteria that have picked up a plasmid, have the ampicillin resistance gene and can grow on an ampicillin containing plate./nNow, how do I get a plasmid with an ampicillin resistance gene? It's in the catalog. It's all there, right? In fact, these occur naturally. You can, with restriction enzymes, move the ampicillin resistance gene to your favorite plasmid./nIf you don't like that, you can put in kanamycin resistance, etc., etc., etc. So, that's how you do it. So, we've got the big picture here. We have now gotten a library, the Library of Human Fragments contained in E coli./nThe library is a big Petri plate or many Petri plates, each one of which is a colony. Each colony has a single vector with an origin, a/nresistance marker, and a distinct piece of human DNA. In this library lives somewhere the gene for Huntington's disease./nOver here is a gene for cystic fibrosis, over here a gene for Duchenne muscular dystrophy, over here a gene for diastrophic dysplasia, over here a gene for etc., etc. The only detail, now, you've got a library./nYou've managed to purify each piece of human DNA away from every other piece of human DNA. The only question now is how do you use the library? How do you go to the library and withdraw the correct volume from the shelf? How do you find the one you're looking for? So, we have converted the problem of purification, which in every other form of biochemistry starts by saying, "I'm going to purify something based on its distinctive properties", to "I'm going to randomly purify everything"./nEverything would be purified in its own bacteria, and I've now converted to the problem of finding the one that I want in my library. Next time, we'll talk about how you go to the library.
Tags // Recombinant DNA Lecture
Added: April 2, 2009, 4:40 am
Runtime: 3010.60 | Views: 26128 | Comments:0

Lecture on molecular biology: introduction to biology
In this video lecture, Central dogma, translation of RNA to proteins and replication of DNA is presented. The lecture was delivered by Professor Eric Lander at MIT. Edited by Ashraf
Tags // molecular biology lecture mit
Added: April 2, 2009, 4:34 am
Runtime: 3082.80 | Views: 18210 | Comments:0