WEBVTT 00:00:03.000 --> 00:00:17.000 Good morning. 00:00:17.000 --> 00:00:23.000 It can't go without at least some acknowledgement dimension. 00:00:23.000 --> 00:00:29.000 If you should ever find yourself in 00:00:29.000 --> 00:00:35.000 life in a situation where you have or are about to give up all hope, 00:00:35.000 --> 00:00:42.000 you think things are utterly impossible and there's no way, 00:00:42.000 --> 00:00:49.000 you will remember this week that nothing is impossible. 00:00:49.000 --> 00:00:53.000 It is possible to come back three games down in the bottom of the 00:00:53.000 --> 00:00:58.000 ninth inning, you've got to believe you can do it, 00:00:58.000 --> 00:01:03.000 and remember to have Dave Roberts pinch run. 00:01:03.000 --> 00:01:06.000 Just a general bit of good advice. What an amazing week, just 00:01:06.000 --> 00:01:09.000 absolutely amazing week. Wow. There are lessons in life to 00:01:09.000 --> 00:01:12.000 be taken from it. Please do take them. 00:01:12.000 --> 00:01:15.000 You know, there really are. I mean I'd given up hope by that 00:01:15.000 --> 00:01:18.000 point, I confess. I wish I could say oh, 00:01:18.000 --> 00:01:22.000 I knew they were going to pull it out, but I didn't. 00:01:22.000 --> 00:01:25.000 And, boy, they pulled it out one game at a time. 00:01:25.000 --> 00:01:28.000 So, all of you think good thoughts this week. This could be a historic 00:01:28.000 --> 00:01:33.000 week, you know, you were here. Anyway, onward. 00:01:33.000 --> 00:01:45.000 We were talking last time about how 00:01:45.000 --> 00:01:49.000 to analyze your clone. The notion of cloning random pieces 00:01:49.000 --> 00:01:54.000 of DNA, identifying your clone within a library, 00:01:54.000 --> 00:01:59.000 purifying the DNA from a clone, doing some preliminary analysis by 00:01:59.000 --> 00:02:03.000 maybe cutting with a restriction enzyme, then sequencing it using 00:02:03.000 --> 00:02:08.000 these techniques that I'd described would allow you to take the clone 00:02:08.000 --> 00:02:13.000 that was, say, able to rescue the yeast that 00:02:13.000 --> 00:02:17.000 couldn't grow without arginine and figure out what its 00:02:17.000 --> 00:02:22.000 DNA sequence was. You could take the clone that you 00:02:22.000 --> 00:02:26.000 had obtained by hybridizing with the DNA sequence corresponding to the 00:02:26.000 --> 00:02:31.000 protein sequence for beta-globin and sequence it and see the beta-globin 00:02:31.000 --> 00:02:36.000 gene sequence perhaps. This is very powerful. 00:02:36.000 --> 00:02:40.000 I want to take a brief moment, we'll come back to it in more detail 00:02:40.000 --> 00:02:44.000 in a subsequent lecture, but I really described how you would 00:02:44.000 --> 00:02:49.000 sequence one clone. I just want to make a note, 00:02:49.000 --> 00:02:53.000 because someone asked about it last time, about how you would sequence 00:02:53.000 --> 00:02:58.000 an entire genome. Someone asked about this. 00:02:58.000 --> 00:03:04.000 Remember before we pulled out our clone, we sequenced it, 00:03:04.000 --> 00:03:10.000 we got its DNA sequence. What if I wanted to sequence the 00:03:10.000 --> 00:03:17.000 entirety of a genome? Yeah. Do a lot of this, 00:03:17.000 --> 00:03:23.000 right, basically if I got a whole genome. Well, 00:03:23.000 --> 00:03:30.000 somebody asked could I put a primer here and just sequence? 00:03:30.000 --> 00:03:34.000 It would take a very long time. And it turns out that it wouldn't 00:03:34.000 --> 00:03:38.000 work because the separation that you can achieve through gels is a 00:03:38.000 --> 00:03:43.000 function, the separation between N and N plus 1 in length goes like the 00:03:43.000 --> 00:03:47.000 logarithm of the ratio. So, it turns out that when N and N 00:03:47.000 --> 00:03:52.000 plus 1 get to like about a thousand, you can achieve very little physical 00:03:52.000 --> 00:03:56.000 separation between them. And so, DNA sequencing runs cannot 00:03:56.000 --> 00:04:01.000 go much past the thousand bases. So, the problem with sequencing a 00:04:01.000 --> 00:04:05.000 genome by putting down a primer on an extraordinarily long piece of DNA, 00:04:05.000 --> 00:04:09.000 a hundred million bases, is you cannot separate the little 00:04:09.000 --> 00:04:14.000 fragments like that. So, what you do is you break up 00:04:14.000 --> 00:04:18.000 your genome into lots of pieces. One strategy, break it up into a 00:04:18.000 --> 00:04:22.000 library of some very big pieces. It turns out you can make pieces at 00:04:22.000 --> 00:04:27.000 random of a hundred thousand base pairs. 00:04:27.000 --> 00:04:31.000 Cloning these in bacterial artificial chromosomes, 00:04:31.000 --> 00:04:36.000 as we talked about before. Take a library of bacterial 00:04:36.000 --> 00:04:40.000 artificial chromosomes and then begin sequencing them. 00:04:40.000 --> 00:04:45.000 And take any given bacterial artificial chromosome and break it 00:04:45.000 --> 00:04:49.000 up into a whole lot of pieces that are maybe a thousand bases long, 00:04:49.000 --> 00:04:54.000 and you could sequence all of those. How do you arrange to get just a 00:04:54.000 --> 00:04:58.000 perfect overlapping set of thousand based pair clones that perfectly 00:04:58.000 --> 00:05:03.000 tile across the sequence with no redundancy? 00:05:03.000 --> 00:05:06.000 You don't. That's the correct answer. That's how you do it, 00:05:06.000 --> 00:05:10.000 you don't. Instead you just randomly take a bunch of things. 00:05:10.000 --> 00:05:13.000 And, in fact, typically you might take clones that give you six or 00:05:13.000 --> 00:05:17.000 eight-fold redundancy. You just sequence a lot of clones 00:05:17.000 --> 00:05:20.000 and then you ask the computer to reassemble it. 00:05:20.000 --> 00:05:24.000 And, in fact, all that overlap is very good for being able to stick 00:05:24.000 --> 00:05:27.000 these pieces together. Sometimes people do such things as 00:05:27.000 --> 00:05:31.000 take pieces that might be four thousand bases long and sequence a 00:05:31.000 --> 00:05:35.000 thousand bases here and a thousand bases here by using a primer that 00:05:35.000 --> 00:05:39.000 starts there and a primer that starts there. And then you can get 00:05:39.000 --> 00:05:42.000 DNA sequences from two ends of a clone. And if you had that for 00:05:42.000 --> 00:05:46.000 zillions of clones your computer program might do an even better job 00:05:46.000 --> 00:05:50.000 of linking things up. It's one very big crossword puzzle 00:05:50.000 --> 00:05:54.000 of putting together all of these pieces, a jigsaw puzzle of putting 00:05:54.000 --> 00:05:58.000 together all these pieces. But, in effect, this is how you 00:05:58.000 --> 00:06:01.000 sequence a big piece of DNA. You chop it up into medium-sized 00:06:01.000 --> 00:06:05.000 pieces of DNA and then tinny pieces of DNA, you sequence them, 00:06:05.000 --> 00:06:09.000 and you use computational science to reassemble it. 00:06:09.000 --> 00:06:13.000 Some people, for some genomes, take the whole big genome and 00:06:13.000 --> 00:06:16.000 immediately go to lots of little pieces. That can work, 00:06:16.000 --> 00:06:20.000 too. I depends on exactly how complicated your genome is. 00:06:20.000 --> 00:06:24.000 In the human genome, there are some parts of a human genome that are 00:06:24.000 --> 00:06:28.000 almost identical that might be like 99.91% identical in two different 00:06:28.000 --> 00:06:32.000 parts of the genome. And so, if you do that, 00:06:32.000 --> 00:06:36.000 you may have trouble telling those pieces apart. So, 00:06:36.000 --> 00:06:40.000 for really complicated genomes people like sometimes breaking it up 00:06:40.000 --> 00:06:45.000 into intermediate-sized pieces. But basically the idea of 00:06:45.000 --> 00:06:49.000 sequencing a big piece of DNA by this process is referred to as 00:06:49.000 --> 00:06:53.000 shotgun sequencing. Shotgun sequencing, 00:06:53.000 --> 00:06:58.000 in fact, was developed in about 1980 by Fred Sanger, 00:06:58.000 --> 00:07:02.000 the same guy who developed the DNA sequencing technique that I told you 00:07:02.000 --> 00:07:07.000 about using polymerase and dideoxynucleotides. 00:07:07.000 --> 00:07:09.000 Sanger very quickly wanted to go from sequencing a single piece to 00:07:09.000 --> 00:07:12.000 sequencing pieces, and so he developed the shotgun 00:07:12.000 --> 00:07:15.000 technique there. And it's now been applied in many 00:07:15.000 --> 00:07:18.000 different forms of intermediate shotguns, whole genome shotguns, 00:07:18.000 --> 00:07:21.000 et cetera. So, that's in reply to the question someone asked last time 00:07:21.000 --> 00:07:24.000 about, well, how would you do a whole genome? And, 00:07:24.000 --> 00:07:27.000 as a matter of fact, this is not theoretical because, 00:07:27.000 --> 00:07:30.000 in fact, people do hold genomes this way. And we do this at MIT. 00:07:30.000 --> 00:07:35.000 Lots of genomes get done here in this fashion. Someone else asked 00:07:35.000 --> 00:07:40.000 how would you analyze your clone. And, again, I'll just make a brief 00:07:40.000 --> 00:07:46.000 remark on that in response to the question. So, 00:07:46.000 --> 00:07:51.000 analyzing some DNA sequence. So, suppose we got some DNA 00:07:51.000 --> 00:07:57.000 sequence, A-A-T-A, don't bother writing this down. 00:07:57.000 --> 00:08:03.000 I'm just making up letters here. How would we make any sense of it? 00:08:03.000 --> 00:08:09.000 Suppose I give you the ones and zeros from your hard-drive, 00:08:09.000 --> 00:08:15.000 how would you make any sense out of them? This is about as interesting 00:08:15.000 --> 00:08:21.000 as the ones as yours from your hard-drive, right? 00:08:21.000 --> 00:08:27.000 It's got four letters, not two, but this is actually what 00:08:27.000 --> 00:08:31.000 you get out of any project. You want to sequence beta-globin? 00:08:31.000 --> 00:08:35.000 You'll get something like this. You want to sequence the arginine 00:08:35.000 --> 00:08:39.000 gene? You want to sequence the human genome? You get a very long 00:08:39.000 --> 00:08:43.000 string of four letters. What do you do with it? 00:08:43.000 --> 00:08:46.000 Oh, well, you could compare it to a normal copy of the gene. 00:08:46.000 --> 00:08:50.000 And if I did that I might find a bunch of differences. 00:08:50.000 --> 00:08:54.000 But how would I even know where the beta-globin gene was within this 00:08:54.000 --> 00:08:58.000 sequence? This clone contains beta-globin. How would I even find 00:08:58.000 --> 00:09:02.000 the exons? Yes? Or whatever? Look at codons. 00:09:02.000 --> 00:09:08.000 So, let's start looking at the codons. This codon here? 00:09:08.000 --> 00:09:13.000 Well, or this, maybe it's this codon here. Sorry? 00:09:13.000 --> 00:09:19.000 Find it. Do you see any start codons here? Oh, 00:09:19.000 --> 00:09:24.000 there's an ATG there. So, maybe that's the start codon or 00:09:24.000 --> 00:09:30.000 maybe not. How often do we expect to find an ATG in some 00:09:30.000 --> 00:09:35.000 reading frame? You know, it could happen fairly 00:09:35.000 --> 00:09:39.000 easily. Also, how do we know it's going this way? 00:09:39.000 --> 00:09:44.000 Maybe we should look for an ATG, we'll put it there, 00:09:44.000 --> 00:09:48.000 going this way. Sorry? I drew the arrow there? 00:09:48.000 --> 00:09:53.000 Well, that's because it's where the sequence started out on my page. 00:09:53.000 --> 00:09:57.000 It doesn't tell me my gene runs that way. Yes? From five 00:09:57.000 --> 00:10:02.000 prime to three prime. Ah, but it's a double-stranded piece 00:10:02.000 --> 00:10:06.000 of DNA. You see, if it's five prime to three prime on 00:10:06.000 --> 00:10:11.000 this strand, the genome has a, another strand that reads the other 00:10:11.000 --> 00:10:16.000 way. What did I get? C-A-T-A, right, C-C-T, 00:10:16.000 --> 00:10:20.000 et cetera. And the gene could be encoded on this strand. 00:10:20.000 --> 00:10:25.000 This could be the coding strand, that could be the coding strand, and 00:10:25.000 --> 00:10:30.000 looking for a mere ATG in one of three possible reading frames on one 00:10:30.000 --> 00:10:35.000 of two possible strands I'll find all sorts of stuff. 00:10:35.000 --> 00:10:38.000 So, sorry? Guess. Guess. Guess is good. 00:10:38.000 --> 00:10:42.000 They don't, don't, won't you, you remember we talked about getting 00:10:42.000 --> 00:10:46.000 papers accepted. If you were to write up the paper 00:10:46.000 --> 00:10:50.000 that way the reviewing would probably ding it and say, 00:10:50.000 --> 00:10:54.000 you know, the guess isn't, isn't good enough. So, that's 00:10:54.000 --> 00:10:58.000 actually very interesting. How do you actually find the gene 00:10:58.000 --> 00:11:02.000 sequence? Well, it turns out to be a 00:11:02.000 --> 00:11:06.000 non-trivial problem which often gets glossed over in the textbooks. 00:11:06.000 --> 00:11:11.000 What you might do is if something really were exonic, 00:11:11.000 --> 00:11:16.000 if this were any exon, does it have any properties that you 00:11:16.000 --> 00:11:20.000 can think of? It shouldn't have a stop codon. No stop codon. 00:11:20.000 --> 00:11:25.000 How often does a stop codon occur at random in a given reading frame? 00:11:25.000 --> 00:11:30.000 How many stop codons are there? Three out of 64 possible codons. 00:11:30.000 --> 00:11:33.000 There's about one in 20 codons in any given reading frame is a stop 00:11:33.000 --> 00:11:37.000 codon. So, that means if I read for about 20 codons, 00:11:37.000 --> 00:11:41.000 and I don't encounter a stop, it's beginning to get more likely 00:11:41.000 --> 00:11:45.000 that that's not random. If I read for, say, 60 codons, 00:11:45.000 --> 00:11:49.000 180 bases and I've encountered no stop codon in that reading frame, 00:11:49.000 --> 00:11:53.000 that chances of that occurring is about either the minus three or so, 00:11:53.000 --> 00:11:57.000 right? Because if I went through three characteristic lengths, 00:11:57.000 --> 00:12:01.000 either the minus three, you know, and I don't know, about 5% or 00:12:01.000 --> 00:12:05.000 something like that. If I went for thousands of bases 00:12:05.000 --> 00:12:09.000 without any stop codon, would you be impressed? That's 00:12:09.000 --> 00:12:14.000 pretty impressive. So, all I have to do is find the 00:12:14.000 --> 00:12:18.000 few thousands of basis with no stop codon. The problem with that is 00:12:18.000 --> 00:12:23.000 that in bacteria there are some genes that are a thousand bases long 00:12:23.000 --> 00:12:27.000 and you, there, you can read them and they have no 00:12:27.000 --> 00:12:33.000 stop codon. What's the problem with the human 00:12:33.000 --> 00:12:39.000 genome? Introns. It turns out that because the 00:12:39.000 --> 00:12:46.000 coding sequences are broken up into small exons, if I found a thousand 00:12:46.000 --> 00:12:53.000 bases with no stop codons then it's very likely coding sequence. 00:12:53.000 --> 00:13:00.000 But a typical human exon is on the order of a 150 to 200 bases. 00:13:00.000 --> 00:13:04.000 Very inconvenient because, you know, it's a typical exon 00:13:04.000 --> 00:13:08.000 encodes 50, 60, 70 codons. So, it turns out that 00:13:08.000 --> 00:13:12.000 even that is not so easy to do. Well, the answer is it's not a 00:13:12.000 --> 00:13:17.000 trivial problem. People do all sorts of things to 00:13:17.000 --> 00:13:21.000 figure out how to decode sequences of genomes. You do run computer 00:13:21.000 --> 00:13:25.000 filters across there that say, look, there are a bunch of 00:13:25.000 --> 00:13:30.000 consecutive codons without stop codons. 00:13:30.000 --> 00:13:33.000 There tend to be little preferences, like amongst the synonymous choices 00:13:33.000 --> 00:13:36.000 of stop codons, humans tend to prefer one stop codon, 00:13:36.000 --> 00:13:39.000 one codon for a specific amino acid over others. So, 00:13:39.000 --> 00:13:43.000 there are some biases as to which codons get used. 00:13:43.000 --> 00:13:46.000 And the computer can kind of take a little bit of account of that. 00:13:46.000 --> 00:13:49.000 Then you can also have made a library of seed DNAs and sequence 00:13:49.000 --> 00:13:53.000 seed DNA, the mRNA which will help you a lot and look for where they 00:13:53.000 --> 00:13:56.000 match up. Then you can take sequences from the human 00:13:56.000 --> 00:13:59.000 and the mouse. And it turns out that the sequences 00:13:59.000 --> 00:14:03.000 in the mouse and the sequences in the human, if you line them up, 00:14:03.000 --> 00:14:06.000 the exons tend to match up better than the introns because evolution 00:14:06.000 --> 00:14:09.000 cares a lot about the exons. But it turns out this is not a 00:14:09.000 --> 00:14:13.000 trivial problem. And even today, if I give you a 00:14:13.000 --> 00:14:16.000 random stretch of human DNA, it's not, there is no simple 00:14:16.000 --> 00:14:20.000 computer program that it's on, that on its own, not even a 00:14:20.000 --> 00:14:23.000 complicated computer program, but on its own would be able, 00:14:23.000 --> 00:14:27.000 without axillary data, to accurately pick out all the genes. 00:14:27.000 --> 00:14:30.000 Even for simple bacteria, we cannot nail perfectly all the 00:14:30.000 --> 00:14:34.000 genes. Although, the lack of these introns means that 00:14:34.000 --> 00:14:38.000 the exons tend to be pretty big, it means the coding are pretty big 00:14:38.000 --> 00:14:42.000 and we can kind of do it. So, I just wanted to point out that, 00:14:42.000 --> 00:14:46.000 that there's a lot still to be done there. The cell manages, 00:14:46.000 --> 00:14:50.000 thank you, to read this just fine, but we're not as smart yet as the 00:14:50.000 --> 00:14:54.000 cell, and so we're not totally able to read out all this stuff. 00:14:54.000 --> 00:14:58.000 We'll come back to genomics in a, in a further lecture. Yes? Yeah, 00:14:58.000 --> 00:15:01.000 wouldn't -- What a cool idea. 00:15:01.000 --> 00:15:04.000 Yes. There are actually some experiments, which maybe if you 00:15:04.000 --> 00:15:08.000 remind me we can, I can work it into a subsequent 00:15:08.000 --> 00:15:11.000 lecture, but people have some experiments where they can randomly 00:15:11.000 --> 00:15:14.000 mutagenize zillions of bacteria and determine which ones will grow and 00:15:14.000 --> 00:15:18.000 which ones won't. And they can do it all in parallel 00:15:18.000 --> 00:15:21.000 in a single test-tube. And thereby you can tell which, 00:15:21.000 --> 00:15:24.000 which nucleotides in the genome matter and which don't. 00:15:24.000 --> 00:15:28.000 It's a kind of cool procedure. OK. Anyway, I just wanted to sort 00:15:28.000 --> 00:15:31.000 of tie up that bit here. Now, let's move on to re-sequencing 00:15:31.000 --> 00:15:35.000 a gene. So, let's suppose we've managed to 00:15:35.000 --> 00:15:39.000 sequence, I don't know, the human genome, the entirety of 00:15:39.000 --> 00:15:43.000 the human genome we have before us, OK? Actually, next week in the 00:15:43.000 --> 00:15:47.000 journal Nature will appear a paper reporting, in fact, 00:15:47.000 --> 00:15:51.000 today, yester, no, yesterday, in fact, it was yesterday, yesterday 00:15:51.000 --> 00:15:55.000 appeared in the journal Nature a paper reporting the finished 00:15:55.000 --> 00:15:59.000 sequence of the human genome. 00:15:59.000 --> 00:16:03.000 http://www.nature. om/nature/journal/v431/n7011/pdf/nature03001.pdf 00:16:03.000 --> 00:16:07.000 And so, anybody who wants to go 00:16:07.000 --> 00:16:11.000 online, in fact, we can get, we'll get copies for the 00:16:11.000 --> 00:16:14.000 class. Why don't we get you copies of the paper? It's not as long as 00:16:14.000 --> 00:16:17.000 the last one. But, in fact, I didn't realize it was 00:16:17.000 --> 00:16:21.000 yesterday. I thought it was next week. Yesterday came out the final 00:16:21.000 --> 00:16:24.000 report on the finished sequence of the human genome, 00:16:24.000 --> 00:16:28.000 which a number of us have been laboring on for quite a long time. 00:16:28.000 --> 00:16:31.000 And it just appeared. So, we actually have that now. 00:16:31.000 --> 00:16:35.000 It actually, it's been on the Web for a while, but the paper 00:16:35.000 --> 00:16:39.000 describing it took a while to write up and it came out yesterday. 00:16:39.000 --> 00:16:43.000 So, we'll get you a copy of that. But now you've got that whole 00:16:43.000 --> 00:16:46.000 sequence of the human genome here. I've been, you know, I've been 00:16:46.000 --> 00:16:50.000 working on this paper with people for so long that, 00:16:50.000 --> 00:16:54.000 you know, I hadn't actually paid attention to the fact that it just 00:16:54.000 --> 00:16:58.000 came out. You don't want to know how long it took to write this. 00:16:58.000 --> 00:17:01.000 The paper, actually, is unusual. It's the only paper that Nature has 00:17:01.000 --> 00:17:05.000 ever published where the author list is sufficiently long that we don't 00:17:05.000 --> 00:17:09.000 have it in the Journal. There's a website that contains the 00:17:09.000 --> 00:17:13.000 author list. There are, I believe, I don't have the final 00:17:13.000 --> 00:17:16.000 count, but something in the neighborhood of about 200 authors to 00:17:16.000 --> 00:17:20.000 the paper. We decided that everybody who'd worked on it should 00:17:20.000 --> 00:17:24.000 be a co-author of the paper, and we just put it all on a website. 00:17:24.000 --> 00:17:28.000 So, anyway, I digress. So, suppose we have the beta-globin gene here. 00:17:28.000 --> 00:17:31.000 So, I've got that in -- I've got the normal form of the 00:17:31.000 --> 00:17:34.000 beta-globin gene, or I've got one person's form, 00:17:34.000 --> 00:17:37.000 in any case, in the human genome sequence. Now I want to take a 00:17:37.000 --> 00:17:41.000 patient with sickle cell anemia and I want to re-sequence their gene. 00:17:41.000 --> 00:17:44.000 Now, remember what we said, we would, we would make a library from 00:17:44.000 --> 00:17:47.000 that person, right? So, we'd get that person's blood, 00:17:47.000 --> 00:17:50.000 we'd purify DNA, we'd cut it, we'd clone it, we'd probe the library 00:17:50.000 --> 00:17:53.000 with a radioactive probe for the beta-globin gene, 00:17:53.000 --> 00:17:56.000 we'd pull out the gene and we'd re-sequence it. 00:17:56.000 --> 00:18:00.000 Suppose we wanted to do that to a hundred patients. 00:18:00.000 --> 00:18:03.000 For every patient we'd get blood, we'd make DNA, we'd clone in a 00:18:03.000 --> 00:18:07.000 plasma, we'd made a whole plasma library, plate it out on filter, 00:18:07.000 --> 00:18:10.000 probe it with a radioactive probe, pull out the clone and sequence it. 00:18:10.000 --> 00:18:14.000 Now, for any such library you probably need to look through a 00:18:14.000 --> 00:18:18.000 couple hundred thousand clones to find beta-globin. 00:18:18.000 --> 00:18:21.000 So, for your DNA and your DNA and your DNA and your DNA, 00:18:21.000 --> 00:18:25.000 we're going to make libraries of a hundred thousand clones, 00:18:25.000 --> 00:18:29.000 that's a couple, that's a lot of plates, right? 00:18:29.000 --> 00:18:32.000 We're going to put them all on, on nylon filters in these 00:18:32.000 --> 00:18:35.000 Seal-a-Meal bags with these radioactive probes, 00:18:35.000 --> 00:18:38.000 and we're going to look for your beta-globin clone, 00:18:38.000 --> 00:18:42.000 your beta-globin clone, your beta-globin clone, your 00:18:42.000 --> 00:18:45.000 beta-globin clone, et cetera, et cetera, 00:18:45.000 --> 00:18:48.000 et cetera. This is really boring. Do you realize how off putting it 00:18:48.000 --> 00:18:51.000 would be to study sickle cell anemia if we had to do that for each 00:18:51.000 --> 00:18:55.000 successive patient, make a whole library? 00:18:55.000 --> 00:18:58.000 But that was what you had to do in molecular biology because that was 00:18:58.000 --> 00:19:02.000 how you got the gene. You build a whole library, 00:19:02.000 --> 00:19:07.000 you withdraw it from the library. However, if you wanted to do this, 00:19:07.000 --> 00:19:12.000 could you manage to get the beta-globin sequence from your 00:19:12.000 --> 00:19:16.000 genome without having to make the whole library? 00:19:16.000 --> 00:19:21.000 It turns out, and I know it's been covered at least in some of the 00:19:21.000 --> 00:19:26.000 sections, there's a cool technique to do that. And what is 00:19:26.000 --> 00:19:32.000 that technique? PCR. So, it turns out that the next 00:19:32.000 --> 00:19:39.000 really great advance in molecular biology was the technique of PCR. 00:19:39.000 --> 00:19:47.000 And what PCR was a way, is, is a way to obtain a piece of DNA 00:19:47.000 --> 00:19:54.000 corresponding to an already known gene, you have to already know the 00:19:54.000 --> 00:20:01.000 gene, and what it allows you to do is then obtain that piece of DNA 00:20:01.000 --> 00:20:09.000 based on knowing at least some of its sequence. 00:20:09.000 --> 00:20:14.000 It allows you to amplify just that DNA from a, from any individual. 00:20:14.000 --> 00:20:20.000 So, as compared to the experiment where I make a library for you and a 00:20:20.000 --> 00:20:25.000 library for you and a library from you and a library from you, 00:20:25.000 --> 00:20:31.000 each of which could take a month, PCR would allow us to do it in 00:20:31.000 --> 00:20:36.000 principle in five minutes. And, actually, 00:20:36.000 --> 00:20:41.000 there are machines that would let you do it in five minutes. 00:20:41.000 --> 00:20:46.000 So, let's discuss how this PCR works. Nobody uses the five minute 00:20:46.000 --> 00:20:52.000 machines because you usually will then wait an hour or so, 00:20:52.000 --> 00:20:57.000 but anyway. Suppose I take my DNA sequence here from 00:20:57.000 --> 00:21:04.000 the human genome. Five prime to three prime. 00:21:04.000 --> 00:21:14.000 Five prime to three prime. This sequence here beta-globin. 00:21:14.000 --> 00:21:23.000 I want to obtain that sequence. The first thing I do is I'm going to 00:21:23.000 --> 00:21:33.000 heat my DNA sample to maybe 97 degrees Celsius to denature. 00:21:33.000 --> 00:21:42.000 Denaturing means, of course, breaking the hydrogen 00:21:42.000 --> 00:21:51.000 bonds that separate the two strands so that the strands come apart, 00:21:51.000 --> 00:22:00.000 five prime to three prime, five prime to three prime. 00:22:00.000 --> 00:22:09.000 Now, what I then do is I take a specific DNA primer matching this 00:22:09.000 --> 00:22:19.000 stretch just before the beta-globin gene starts. 00:22:19.000 --> 00:22:22.000 Or just before where I'm interested in. How do I make a primer that 00:22:22.000 --> 00:22:26.000 matches just that sequence? I order, well, how do I know what 00:22:26.000 --> 00:22:30.000 to order? I know the sequence, right? 00:22:30.000 --> 00:22:34.000 I've got the sequence already. I just look at it and I say I want 00:22:34.000 --> 00:22:38.000 that sequence. And then how do I get it? 00:22:38.000 --> 00:22:42.000 I order it. I type it into the Web and the machine will synthesize me 00:22:42.000 --> 00:22:47.000 this, this primer. Typically a 20-base stretch will 00:22:47.000 --> 00:22:51.000 suffice. So, I'll get me a twentymer, a 20 base oligonucleotide 00:22:51.000 --> 00:22:55.000 complimentary to the sequence on this side of the gene. 00:22:55.000 --> 00:23:00.000 What I'm also going to do is the same thing over here. 00:23:00.000 --> 00:23:06.000 I'm going to get a second primer. This is primer number one. This is 00:23:06.000 --> 00:23:12.000 primer number two. OK? Now, let's see. 00:23:12.000 --> 00:23:19.000 Five prime. This is five prime, five prime. Now what I'd like to do 00:23:19.000 --> 00:23:25.000 is add polymerase, I'd like to add dNTPs. 00:23:25.000 --> 00:23:32.000 So, plus DNA polymerase plus dNTPs. 00:23:32.000 --> 00:23:36.000 And what will happen? Polymerase will come along and 00:23:36.000 --> 00:23:41.000 start copying my DNA, but it will only copy it starting 00:23:41.000 --> 00:23:45.000 from the primers. Now, this will keep going, 00:23:45.000 --> 00:23:50.000 of course, but DNA polymerase doesn't go forever, 00:23:50.000 --> 00:23:55.000 you know, the reactions sort of stops at some point. 00:23:55.000 --> 00:23:59.000 And so you'll get a strand going off here and a strand 00:23:59.000 --> 00:24:04.000 going off there. Now, notice what I've done. 00:24:04.000 --> 00:24:10.000 I started with an entire human genome, and the number of copies of 00:24:10.000 --> 00:24:15.000 beta-globin was one per genome. When I'm done with this process, 00:24:15.000 --> 00:24:21.000 how many copies, how many double-stranded copies of 00:24:21.000 --> 00:24:27.000 beta-globin do I have? Two. That's still very little, 00:24:27.000 --> 00:24:33.000 but it's more than I had before. So, what do I do next? 00:24:33.000 --> 00:24:41.000 Repeat. So, let's heat up that sample again. We'll denature at 97 00:24:41.000 --> 00:24:48.000 degrees, and now we have our initial strand here, we have our strand that 00:24:48.000 --> 00:24:56.000 came off this primer that runs to here and maybe goes forward, 00:24:56.000 --> 00:25:05.000 we have this strand here. We have this strand here. 00:25:05.000 --> 00:25:15.000 And this was five prime, five prime, five prime, five prime. 00:25:15.000 --> 00:25:25.000 Now what do we do? We repeat. We'll take our primer, this is 00:25:25.000 --> 00:25:33.000 primer number one, let's see. It matches over there. 00:25:33.000 --> 00:25:41.000 Primer number two over here. Number one over here. Number two. 00:25:41.000 --> 00:25:48.000 Have I got this right? Yes. Good. Then where does this guy stop? 00:25:48.000 --> 00:25:56.000 Right at the end where my other primer was. This guy 00:25:56.000 --> 00:26:02.000 runs along here. That guy stops right at the end. 00:26:02.000 --> 00:26:07.000 That guy might go a little further. How many copies of the beta-globin 00:26:07.000 --> 00:26:12.000 gene do I have now? Four. Two of which, 00:26:12.000 --> 00:26:17.000 by the way, perfectly sit between my pink primers. What's going to 00:26:17.000 --> 00:26:22.000 happen if I do this again? How many copies will I get? 00:26:22.000 --> 00:26:27.000 Eight, six of which will sit perfectly and two might be a little 00:26:27.000 --> 00:26:32.000 ragged as to where they go. So, initially, 00:26:32.000 --> 00:26:38.000 after cycle number zero, that is initial conditions, 00:26:38.000 --> 00:26:44.000 the number of copies relative to the genome was one. 00:26:44.000 --> 00:26:50.000 After one cycle it's two. After two cycles it's four. 00:26:50.000 --> 00:26:56.000 After N cycles it's two to the N copies. Is that clear 00:26:56.000 --> 00:27:01.000 how the PCR works? And that on every round you're 00:27:01.000 --> 00:27:07.000 doubling. And, with the exception of those two 00:27:07.000 --> 00:27:13.000 white things that go off to the side, they're going back and forth and 00:27:13.000 --> 00:27:18.000 back and forth between the two primers you chose to put in. 00:27:18.000 --> 00:27:24.000 What is when N equals ten, what do you got? A thousand copies. 00:27:24.000 --> 00:27:30.000 What happens when N equals 20? A million copies. 00:27:30.000 --> 00:27:34.000 What is the copy number of beta-globin? Beta, 00:27:34.000 --> 00:27:38.000 let's suppose beta-globin, for the sake of the argument, 00:27:38.000 --> 00:27:42.000 sake of argument is about one thousand bases. 00:27:42.000 --> 00:27:46.000 What fraction of the human genome does beta-globin represent? 00:27:46.000 --> 00:27:50.000 Yeah, about a millionth for the genome. No, actually, 00:27:50.000 --> 00:27:54.000 one three millionth, but we'll call it a millionth. 00:27:54.000 --> 00:27:58.000 So, after I've made a million-fold amplification of beta-globin, 00:27:58.000 --> 00:28:03.000 beta-globin now represents half of the stuff that's in the tube. 00:28:03.000 --> 00:28:08.000 What would happen if I go another ten rounds? How many copies do I 00:28:08.000 --> 00:28:13.000 have? A billion copies. So, in other words, I started with 00:28:13.000 --> 00:28:18.000 something that was only present at about one one-millionth of what was 00:28:18.000 --> 00:28:23.000 in my test tube. If I could make a billion copies of 00:28:23.000 --> 00:28:28.000 that specific molecule, now it so dominates the mixture that 00:28:28.000 --> 00:28:33.000 it is a thousand times more abundant than the rest of the genome. 00:28:33.000 --> 00:28:39.000 It works. That's the remarkable thing, this works. 00:28:39.000 --> 00:28:45.000 Any questions about the technique? Now, yes? Well, I need two primers 00:28:45.000 --> 00:28:52.000 in their sequence. How many copies do I need of each 00:28:52.000 --> 00:28:58.000 of those primers? Well, I, I obviously need a lot of 00:28:58.000 --> 00:29:03.000 copies of those primers. So, primer number one, 00:29:03.000 --> 00:29:06.000 it's a single sequence, but when I order it from the company, 00:29:06.000 --> 00:29:10.000 I'm going to order me a boat load, a lot of that primer. So, I'm 00:29:10.000 --> 00:29:13.000 adding, I better add a billion molecules of that primer because I'm 00:29:13.000 --> 00:29:16.000 going to make a billion copies starting from such primers. 00:29:16.000 --> 00:29:20.000 But if I have a billion copies of, of number one and a billion copies 00:29:20.000 --> 00:29:23.000 of number two and, you know, these days, 00:29:23.000 --> 00:29:26.000 billions aren't such big, you know, molecules are Avogadro's 00:29:26.000 --> 00:29:30.000 number and all that. It's not hard to get things. 00:29:30.000 --> 00:29:34.000 So, you throw in huge excess, a massive excess of primer number 00:29:34.000 --> 00:29:38.000 one, a massive excess of primer number two, and you just do this. 00:29:38.000 --> 00:29:42.000 Now, I mean, what does it cost to make such a massive excess of a 00:29:42.000 --> 00:29:46.000 primer? It's about ten cents a base, so it's two bucks, 00:29:46.000 --> 00:29:50.000 two bucks per primer give or take. You know, so I can get you a better 00:29:50.000 --> 00:29:54.000 price if you want, but, you know, anyway. 00:29:54.000 --> 00:29:58.000 It's not a bad price to, to buy primer. So, you can just go 00:29:58.000 --> 00:30:01.000 out and order a pair of primers. You can have them tomorrow. 00:30:01.000 --> 00:30:05.000 And then all you have to do is add the primers to the, 00:30:05.000 --> 00:30:09.000 so I take DNA. Do I, I, I need DNA from you. 00:30:09.000 --> 00:30:13.000 It turns out I could draw your blood and purify DNA and all that. 00:30:13.000 --> 00:30:16.000 But it turns out that if all I wanted to do was amplify one locus, 00:30:16.000 --> 00:30:20.000 I could actually take a Popsicle stick and ask you to scrape the 00:30:20.000 --> 00:30:24.000 inside of your cheek. That'll get enough cells off from 00:30:24.000 --> 00:30:28.000 the inside of your cheek, stick it in a test-tube, and it'll 00:30:28.000 --> 00:30:31.000 actually have enough DNA there. It turns out this is a very 00:30:31.000 --> 00:30:35.000 sensitive and powerful technique, so, but before we get to that notice 00:30:35.000 --> 00:30:39.000 what we had to do. We had to heat our DNA to 97 00:30:39.000 --> 00:30:43.000 degrees and add polymerase. Then we heat again to 97, add 00:30:43.000 --> 00:30:46.000 polymerase, heat again to 97, add polymerase. Why do I have to 00:30:46.000 --> 00:30:50.000 keep adding polymerase? Because polymerase gets roomed at 00:30:50.000 --> 00:30:54.000 97 degrees so it's denatured. So, the nuisance about PCR is I 00:30:54.000 --> 00:30:57.000 have to go to my Eppendorf plastic tube, pop open the lid, 00:30:57.000 --> 00:31:01.000 stick in some DNA polymerase, close it up, stick it back in a 00:31:01.000 --> 00:31:05.000 heating bath, let it go for a while, take it out, pop it open, add some 00:31:05.000 --> 00:31:09.000 more polymerase, put it back in the heating bath, 00:31:09.000 --> 00:31:13.000 pop it out. And this is actually the way 00:31:13.000 --> 00:31:17.000 primitive scientists did PCR not so long ago, OK? Wouldn't it be cool 00:31:17.000 --> 00:31:22.000 if we could engineer a DNA polymerase that didn't denature at 00:31:22.000 --> 00:31:26.000 97 degrees? Because then what we could do is just add the polymerase, 00:31:26.000 --> 00:31:31.000 close up the tube, put it in a machine that goes heat, 00:31:31.000 --> 00:31:35.000 cool, heat, cool, heat, cool, heat, cool, but you would have, 00:31:35.000 --> 00:31:40.000 so how do we, what kind of cleaver biological engineering do we use to 00:31:40.000 --> 00:31:45.000 modify polymerase so it won't denature at 97 degrees? 00:31:45.000 --> 00:31:49.000 Yes? Get it from a bacteria. What kind of a bacteria would you 00:31:49.000 --> 00:31:53.000 ask for an enzyme that could work in, in basically boiling water? 00:31:53.000 --> 00:31:57.000 Bacteria that basically live in boiling water. Where would 00:31:57.000 --> 00:32:03.000 you look for such? Thermal vents. 00:32:03.000 --> 00:32:09.000 You'll, geysers, things like that. Life lives 00:32:09.000 --> 00:32:15.000 everywhere. What you go is you find yourself a bacterium, 00:32:15.000 --> 00:32:21.000 so you find bacteria that lived in geysers or in thermal vents and you 00:32:21.000 --> 00:32:27.000 purify their DNA polymerase. The most famous one comes from the 00:32:27.000 --> 00:32:34.000 organism, the bacterium called thermos aquaticus, aquaticus. 00:32:34.000 --> 00:32:38.000 Which of course means hot water, right? That's what the bacteria is 00:32:38.000 --> 00:32:43.000 called, thermos aquaticus. And, or, and its enzyme is called 00:32:43.000 --> 00:32:47.000 tack, tack. So, we'll refer to it often, 00:32:47.000 --> 00:32:52.000 Taq polymerase, meaning from this bacteria thermos aquaticus, 00:32:52.000 --> 00:32:57.000 OK? So, that's Taq. So, it turns out that you can do this now without 00:32:57.000 --> 00:33:02.000 having to open and close the test-tubes. 00:33:02.000 --> 00:33:11.000 Oops, I meant to put that here. How sensitive is PCR? It's very 00:33:11.000 --> 00:33:20.000 sensitive. You could do, so applications of PCR. Very 00:33:20.000 --> 00:33:30.000 versatile. First let's just re-sequence a gene. 00:33:30.000 --> 00:33:36.000 Gene from yeast or from human. You just need, you know, any DNA 00:33:36.000 --> 00:33:42.000 sample. Get my gene, get my primers, and as I was 00:33:42.000 --> 00:33:48.000 indicating with a Popsicle stick, I don't have to have it very pure, 00:33:48.000 --> 00:33:54.000 although in a laboratory you go to the trouble of making it pure 00:33:54.000 --> 00:34:00.000 because you want it to be pure and all that. Yes? 00:34:00.000 --> 00:34:06.000 Correct. Yeah. So, remember I was making a fuss 00:34:06.000 --> 00:34:12.000 over the accuracy of replication, right? And I said that on its own a 00:34:12.000 --> 00:34:19.000 polymerase might have an accuracy to only about ten to the minus five. 00:34:19.000 --> 00:34:25.000 So, now, what were the two mechanisms for, 00:34:25.000 --> 00:34:32.000 for repairing DNA, for proofreading DNA? 00:34:32.000 --> 00:34:35.000 One was a built-in proofreading activity that the enzyme had. 00:34:35.000 --> 00:34:38.000 The enzyme would have put in a base, would check the base, 00:34:38.000 --> 00:34:42.000 and that actually helped by an order of magnitude or two. 00:34:42.000 --> 00:34:45.000 And some of these polymerases have a proofreading activity. 00:34:45.000 --> 00:34:49.000 But then we also discussed the mismatch repair activity that would 00:34:49.000 --> 00:34:52.000 later come along and detect mismatches. You're absolutely right, 00:34:52.000 --> 00:34:56.000 PCR is not as accurate as cells because it doesn't have that 00:34:56.000 --> 00:35:00.000 mismatch repair activity. So, when you take a PCR product, 00:35:00.000 --> 00:35:05.000 if I were to clone, so if I were to take all the PCR product, 00:35:05.000 --> 00:35:10.000 say, from my beta-globin gene, so I'm going to take my test-tube, 00:35:10.000 --> 00:35:15.000 I'm going to add my primers and everything, I'm going to PCR, 00:35:15.000 --> 00:35:20.000 I'm going to PCR, and then I'm going to get a lot of copies of 00:35:20.000 --> 00:35:25.000 beta-globin. If I were to take that beta-globin and just directly 00:35:25.000 --> 00:35:30.000 sequence the DNA in the test-tube. Here's my pieces of beta-globin. 00:35:30.000 --> 00:35:34.000 I can now sequence it by adding a primer and doing my fluorescent 00:35:34.000 --> 00:35:39.000 sequencing and running it on a sequencer and all that. 00:35:39.000 --> 00:35:44.000 Sorry, going the other way. I'll run a sequencing reaction. 00:35:44.000 --> 00:35:48.000 I could actually do it, and what I do it on is the whole population of 00:35:48.000 --> 00:35:53.000 a million or a billion molecules. If any one of them is wrong it's 00:35:53.000 --> 00:35:58.000 going to be swamped out by others, OK? Because I could do my 00:35:58.000 --> 00:36:03.000 sequencing reaction on the whole PCR product. 00:36:03.000 --> 00:36:07.000 And random mistakes in one molecule or the other will still be a tiny 00:36:07.000 --> 00:36:12.000 minority of the votes at any given base, right? But suppose I were to 00:36:12.000 --> 00:36:17.000 take my PCR product, all these amplified molecules here, 00:36:17.000 --> 00:36:22.000 and suppose I were to clone them individually and I were to sequence 00:36:22.000 --> 00:36:27.000 each of those individual clones instead of sequencing a, 00:36:27.000 --> 00:36:32.000 a mixture of all the products. I would, in fact, 00:36:32.000 --> 00:36:36.000 see a higher mutation rate. And you're absolutely right. 00:36:36.000 --> 00:36:41.000 When people clone PCR products they have to check them afterwards and 00:36:41.000 --> 00:36:46.000 throw out the ones that are wrong, OK? Absolutely right. Good, good, 00:36:46.000 --> 00:36:50.000 good. So, you guys are, you know, right on top of the important issues 00:36:50.000 --> 00:36:55.000 about, about DNA. So, so I can, I can take a gene and 00:36:55.000 --> 00:37:00.000 I can re-sequence it. I can also do things like take 00:37:00.000 --> 00:37:05.000 blood and look for the presence of a virus. 00:37:05.000 --> 00:37:11.000 So, I could re-sequence beta-globin and study people and see who's got 00:37:11.000 --> 00:37:18.000 sickle cell anemia and all that. I could take blood and I might want 00:37:18.000 --> 00:37:24.000 to say do I see the HIV virus present in someone's blood? 00:37:24.000 --> 00:37:31.000 For example, HIV testing can be done by making PCR primers for the 00:37:31.000 --> 00:37:37.000 sequence of the HIV virus. It has a genome. 00:37:37.000 --> 00:37:41.000 Taking a human's blood sample and PCR-ing it. If you get a positive 00:37:41.000 --> 00:37:45.000 PCR product, a PCR product that is made by these two primers and if, 00:37:45.000 --> 00:37:49.000 for example, you checked that it, that it gives you the HIV sequence 00:37:49.000 --> 00:37:53.000 then you know that that blood sample has, that person has the HIV virus. 00:37:53.000 --> 00:37:57.000 This is a way to do this. The PCR reaction itself is fast. 00:37:57.000 --> 00:38:01.000 Typically takes hours. In fact, can be forced to go much 00:38:01.000 --> 00:38:04.000 more quickly by machines that rapidly thermocycle. 00:38:04.000 --> 00:38:08.000 And you can actually PCR in five minutes, although people don't do it 00:38:08.000 --> 00:38:11.000 very often, but if you put a thin glass capillary and go heat, 00:38:11.000 --> 00:38:14.000 cold, heat, cold very, very quickly, there's a machine from Idaho 00:38:14.000 --> 00:38:18.000 Technologies that can do it in five minutes, but it's usually not the 00:38:18.000 --> 00:38:21.000 trouble. And you just put it in and, you know, in a couple hours you'll 00:38:21.000 --> 00:38:24.000 get an answer there as to whether or not somebody has HIV, 00:38:24.000 --> 00:38:28.000 for example. So, you can do that to detect relatively low 00:38:28.000 --> 00:38:33.000 quantities of virus. How low can you go? 00:38:33.000 --> 00:38:39.000 Well, it turns out, what's the limit? What's the 00:38:39.000 --> 00:38:45.000 smallest number of molecules you might be able to detect in a sample? 00:38:45.000 --> 00:38:51.000 Theoretically. One. You can't fewer than one molecule, 00:38:51.000 --> 00:38:57.000 right? So, one might be the limit. So, how could I arrange to have a 00:38:57.000 --> 00:39:01.000 single molecule in a test-tube? I would like to have a test-tube 00:39:01.000 --> 00:39:04.000 that has exactly one copy of the beta-globin gene. 00:39:04.000 --> 00:39:08.000 What, how's the best, what's the best way to get exactly 00:39:08.000 --> 00:39:11.000 one copy of beta-globin and put it in the test-tube? 00:39:11.000 --> 00:39:14.000 Sorry? You can't. Why? Just one molecule. 00:39:14.000 --> 00:39:18.000 I want to get exactly one copy of beta-globin. I could, 00:39:18.000 --> 00:39:21.000 I could just take total DNA and dilute it so, on average, 00:39:21.000 --> 00:39:24.000 there's only one copy. Or, actually, is there any way to, 00:39:24.000 --> 00:39:28.000 I mean can I, I'd just like to buy a package that contains exactly 00:39:28.000 --> 00:39:32.000 one beta-globin. Sorry? Bind it to something big. 00:39:32.000 --> 00:39:36.000 Let's think biologically. Does biology package up a single copy of 00:39:36.000 --> 00:39:41.000 beta-globin? Sorry? Gametes. How about a sperm? 00:39:41.000 --> 00:39:46.000 Let's grab a sperm by its tail here, put it in the test-tube. 00:39:46.000 --> 00:39:50.000 It's one copy of beta-globin. So, you can actually take cell 00:39:50.000 --> 00:39:55.000 sorters and have it cell sort sperm into individual test-tubes. 00:39:55.000 --> 00:40:00.000 You now know there's one copy of beta-globin. 00:40:00.000 --> 00:40:05.000 Heat it up, it will crack open the sperm, add your primers, 00:40:05.000 --> 00:40:10.000 you can amplify beta-globin, it's a single copy. That proves its 00:40:10.000 --> 00:40:16.000 extraordinary sensitivity. You can do it with a single sperm. 00:40:16.000 --> 00:40:21.000 You can do it with a single egg also, but harder to come by. 00:40:21.000 --> 00:40:27.000 So, with that level of sensitivity, you could do the following. So, 00:40:27.000 --> 00:40:32.000 single sperm typing. Now, single sperm typing is cool but 00:40:32.000 --> 00:40:36.000 sort of useless. What are you going to do with it, 00:40:36.000 --> 00:40:41.000 right? But here's another thing you could do. Embryo typing. 00:40:41.000 --> 00:40:45.000 Suppose someone has a genetic disease in their family, 00:40:45.000 --> 00:40:50.000 maybe it's Huntington's disease. And suppose that the individual with 00:40:50.000 --> 00:40:54.000 Huntington's disease wants to have kids. Or the individual, 00:40:54.000 --> 00:40:59.000 sorry, the individual who is at risk for Huntington's disease or breast 00:40:59.000 --> 00:41:04.000 cancer or whatever wants to have kids. 00:41:04.000 --> 00:41:13.000 What you can do is with an in vitro fertilization clinic you're able to 00:41:13.000 --> 00:41:23.000 obtain eggs, fertilize eggs in vitro, and grow them up in a Petri plate to 00:41:23.000 --> 00:41:33.000 8 or 16 cell stage before re-implanting embryos 00:41:33.000 --> 00:41:41.000 back in the mother. Wouldn't it be cool if we could 00:41:41.000 --> 00:41:48.000 choose to only re-implant an embryo that did not have the genetic 00:41:48.000 --> 00:41:56.000 disease? How are we going to do that? PCR. How are we, 00:41:56.000 --> 00:42:02.000 so what do we do? We take the embryo. 00:42:02.000 --> 00:42:06.000 We make DNA from the embryo. We do PCR and we say, ah-ha, this 00:42:06.000 --> 00:42:10.000 embryo did not have the genetic disease. Problem is it has killed 00:42:10.000 --> 00:42:15.000 the, the cells there, right, it killed the embryo. 00:42:15.000 --> 00:42:19.000 Any ideas? Pull off one cell. Remove a single cell. It turns out 00:42:19.000 --> 00:42:24.000 that at stage the cells are not differentiated. 00:42:24.000 --> 00:42:28.000 If I remove one cell from an embryo at that very early stage, 00:42:28.000 --> 00:42:33.000 the other cells with make a perfectly happy, healthy baby. 00:42:33.000 --> 00:42:37.000 That cell is not necessary. This single cell sensitivity is 00:42:37.000 --> 00:42:41.000 very valuable because I can actually do single cell genotyping on in 00:42:41.000 --> 00:42:45.000 vitro fertilized embryos and be able offer parents a chance, 00:42:45.000 --> 00:42:49.000 the opportunity to re-implant only those embryos that do not have the 00:42:49.000 --> 00:42:53.000 genetic defect. That's cool. That's really cool. 00:42:53.000 --> 00:42:57.000 There are other things you might be able to do. If you're treating a 00:42:57.000 --> 00:43:01.000 patient with cancer, a patient, a cancer patient and 00:43:01.000 --> 00:43:05.000 you've given chemotherapy you want to know have I managed to eradicate 00:43:05.000 --> 00:43:11.000 the cancer cells? And six months later have any of the 00:43:11.000 --> 00:43:19.000 cancer cells come back? I could look for very low 00:43:19.000 --> 00:43:27.000 quantities of cancer cells. I can, I can do surveillance for 00:43:27.000 --> 00:43:35.000 low quantities of cancer cells following chemotherapy. 00:43:35.000 --> 00:43:39.000 And, of course, I can also do forensics. 00:43:39.000 --> 00:43:44.000 I could take a small sample of blood from the scene of a crime or 00:43:44.000 --> 00:43:49.000 saliva from the back of an envelope that someone has licked, 00:43:49.000 --> 00:43:53.000 and I could do PCR and look for genetic variations that distinguish 00:43:53.000 --> 00:43:58.000 people. And, presumably, you see all that stuff on television 00:43:58.000 --> 00:44:04.000 all the time. So, that's what PCR is good for. 00:44:04.000 --> 00:44:10.000 It's good. All right. Last topic, very brief topic, but I do want to 00:44:10.000 --> 00:44:17.000 mention. This was being able to analyze a gene directed mutagenesis. 00:44:17.000 --> 00:44:23.000 And I won't go through the details of all this, but I just want to at 00:44:23.000 --> 00:44:30.000 least basically describe the concept. 00:44:30.000 --> 00:44:36.000 I could take any piece of DNA, say from a drosophila, and I can 00:44:36.000 --> 00:44:42.000 mutate the DNA in vitro. I can change this base from a G to 00:44:42.000 --> 00:44:48.000 a C. There's a right, there's a proper protocol and 00:44:48.000 --> 00:44:54.000 cooking trick for doing that. It involves putting a certain oligo 00:44:54.000 --> 00:45:00.000 over it and extending, and it doesn't matter exactly how. 00:45:00.000 --> 00:45:04.000 I could insert an extra gene into that. I could use a little 00:45:04.000 --> 00:45:08.000 restriction enzyme to open it up and stuff something in. 00:45:08.000 --> 00:45:13.000 I could delete something from this. Maybe I'll use a restriction enzyme 00:45:13.000 --> 00:45:17.000 to cut it open, et cetera. Basically I could, 00:45:17.000 --> 00:45:22.000 I can fuse genes together. I can do whatever kind of construction of 00:45:22.000 --> 00:45:26.000 pieces of DNA and modifications of pieces of DNA that I would 00:45:26.000 --> 00:45:31.000 like to do in vitro. I can then take that mutated gene, 00:45:31.000 --> 00:45:36.000 let's say the gene is an enzyme, encodes an enzyme, 00:45:36.000 --> 00:45:41.000 and the enzyme has an active site. I could change the code for the 00:45:41.000 --> 00:45:46.000 amino acid right at the active site to see if that amino acid really 00:45:46.000 --> 00:45:52.000 matters or not. I can do any of those things. 00:45:52.000 --> 00:45:57.000 And I can put this back in an organism. Remember that you, 00:45:57.000 --> 00:46:02.000 I said you could transform DNA back into bacteria? 00:46:02.000 --> 00:46:07.000 Well, you can also do such things as simply inject DNA into 00:46:07.000 --> 00:46:11.000 a fertilized egg. In fact, at the stage where there's 00:46:11.000 --> 00:46:15.000 a male and a female pronucleus that haven't fused yet right after 00:46:15.000 --> 00:46:19.000 fertilization. You can take your little pipette 00:46:19.000 --> 00:46:22.000 and a needle and you can inject some of the DNA you want into the male 00:46:22.000 --> 00:46:26.000 pronucleus, and then when the male pronucleus and the female pronucleus 00:46:26.000 --> 00:46:30.000 fuse and the embryo grows it will have your DNA. 00:46:30.000 --> 00:46:36.000 You can make mice that carry whatever gene you've modified like 00:46:36.000 --> 00:46:42.000 this. You can also not, you, you can also not just modify a 00:46:42.000 --> 00:46:49.000 piece of DNA and add, this is gene addition, 00:46:49.000 --> 00:46:55.000 you can also do gene subtraction. You can do gene subtraction and, 00:46:55.000 --> 00:47:01.000 again, I won't worry about the details here, by taking 00:47:01.000 --> 00:47:07.000 embryonic stem cells. Much in the news these days, 00:47:07.000 --> 00:47:11.000 and we may come back to them. And in vitro, working with 00:47:11.000 --> 00:47:15.000 embryonic stem cells, to transform a piece of DNA that has 00:47:15.000 --> 00:47:19.000 been arranged to recombine into the gene of interest and know it out. 00:47:19.000 --> 00:47:24.000 So, if you build, if you build a piece of DNA in vitro and you put it 00:47:24.000 --> 00:47:28.000 into a whole bunch of embryonic stem cells you can select, 00:47:28.000 --> 00:47:32.000 by various cleaver techniques, for those embryonic stem cells that 00:47:32.000 --> 00:47:38.000 have taken up your gene. And not just taken it up but slammed 00:47:38.000 --> 00:47:45.000 it into the normal locus in place of the normal locus. 00:47:45.000 --> 00:47:52.000 And that way you can knock out a gene. You can do gene knockout. 00:47:52.000 --> 00:47:59.000 So, the basic point of this now, to summarize these many lectures is 00:47:59.000 --> 00:48:06.000 we're now at the point where this picture that we saw at the, 00:48:06.000 --> 00:48:13.000 at the beginning, function, gene, protein, that we understood now 00:48:13.000 --> 00:48:20.000 first as a methodology, genetics, biochemistry. 00:48:20.000 --> 00:48:26.000 And then we understood how genes encode proteins through molecular 00:48:26.000 --> 00:48:32.000 biology. These tools of recombinant DNA allow us to move 00:48:32.000 --> 00:48:37.000 in any direction. You want to find the gene underlying 00:48:37.000 --> 00:48:41.000 a function, find the gene for Huntington's disease? 00:48:41.000 --> 00:48:45.000 We could do it. Clone it based solely on its linkage. 00:48:45.000 --> 00:48:49.000 You want to find the gene encoding a protein? If I know its amino acid 00:48:49.000 --> 00:48:53.000 sequence, I can find the DNA sequence that corresponds it. 00:48:53.000 --> 00:48:57.000 If I want to find what a certain protein does, its function, 00:48:57.000 --> 00:49:01.000 I could get the gene for that protein. I could knock out the gene 00:49:01.000 --> 00:49:05.000 for that protein and see what its function is. 00:49:05.000 --> 00:49:08.000 Suddenly, for the mathematicians amongst the group, 00:49:08.000 --> 00:49:12.000 this becomes a commutative diagram, which you can chase around in any 00:49:12.000 --> 00:49:15.000 direction. That is, in a sense, what the 20th century 00:49:15.000 --> 00:49:19.000 was about, was intellectually these two disciplines merging through 00:49:19.000 --> 00:49:22.000 molecular biology and then recombinant DNA giving you all the 00:49:22.000 --> 00:49:26.000 tools that if you're sitting at any place in this triangle you can move 00:49:26.000 --> 00:49:29.000 this way and that way, from a gene to a protein, 00:49:29.000 --> 00:49:33.000 from a protein to a gene, from a function to a gene, from a 00:49:33.000 --> 00:49:36.000 function to a protein. Much of the rest of the course we'll 00:49:36.000 --> 00:49:40.000 talk about how you use these tools, but this brings to a close this 00:49:40.000 --> 00:49:44.000 first chunk of the course about the concepts and the methodologies of 00:49:44.000 --> 00:49:48.000 molecular biology. Now, if you hang on one more minute, 00:49:48.000 --> 00:49:52.000 this is my last lecture for a while. I won't be, we're having an exam on, 00:49:52.000 --> 00:49:56.000 we have a quiz on Monday, and then Bob's taking over again. 00:49:56.000 --> 00:50:00.000 So, I won't see you for the next week or so. So, two things. 00:50:00.000 --> 00:50:04.000 One, I won't see you before the World Series is over so everyone 00:50:04.000 --> 00:50:09.000 please think good thoughts about the Red Sox. Number two, 00:50:09.000 --> 00:50:13.000 I will not see you before the election. Vote. 00:50:13.000 --> 00:50:18.000 It's your choice who you vote for, but vote. Good-bye.