WEBVTT 00:00:16.750 --> 00:00:20.470 ADAM MARTIN: All right, so we're going to switch gears again 00:00:20.470 --> 00:00:25.060 today, and we're going to move off of kind of pure genetics 00:00:25.060 --> 00:00:28.150 and start to talk about molecular genetics. 00:00:28.150 --> 00:00:32.650 And I want to start with the concept of-- let's say 00:00:32.650 --> 00:00:36.490 you want to identify a piece of DNA, 00:00:36.490 --> 00:00:40.210 purify it, and propagate it so that you have it 00:00:40.210 --> 00:00:43.000 for future use. 00:00:43.000 --> 00:00:49.360 And so the process of doing this is known as cloning. 00:00:49.360 --> 00:00:52.750 And it's the process of, if you will, 00:00:52.750 --> 00:00:59.140 purifying and propagating a piece of DNA in an organism. 00:01:07.900 --> 00:01:10.600 So sort of the goal for this lecture 00:01:10.600 --> 00:01:14.530 is for you to know how if you wanted to, let's say, 00:01:14.530 --> 00:01:16.690 identify a piece of DNA-- 00:01:16.690 --> 00:01:18.850 maybe you're interested in the piece of DNA 00:01:18.850 --> 00:01:20.860 that contains a given gene. 00:01:20.860 --> 00:01:26.170 How would you go about getting that DNA in a state that can be 00:01:26.170 --> 00:01:29.050 propagated sort of on and on? 00:01:29.050 --> 00:01:31.930 And how can you identify the piece of DNA you want? 00:01:34.510 --> 00:01:37.270 And so one tool that we're going to use 00:01:37.270 --> 00:01:43.010 is we're going to use an organism bacteria as a tool. 00:01:43.010 --> 00:01:48.070 So I'll draw my sample bacteria cell here. 00:01:48.070 --> 00:01:51.670 Could be something like E. coli, just some bacterium. 00:01:56.620 --> 00:02:03.280 And you'll recall, when I talked about cells a few weeks ago, 00:02:03.280 --> 00:02:06.580 bacteria and prokaryotic cells have 00:02:06.580 --> 00:02:11.560 a chromosome called the nucleoid that's present inside. 00:02:11.560 --> 00:02:13.420 So that's the bacterial chromosome. 00:02:16.780 --> 00:02:21.760 But bacteria can also have extra chromosomal pieces of DNA 00:02:21.760 --> 00:02:26.308 that are called plasmids that exist in their cytoplasm. 00:02:26.308 --> 00:02:27.100 These are plasmids. 00:02:30.760 --> 00:02:34.360 And these extra chromosomal pieces of DNA 00:02:34.360 --> 00:02:38.440 replicate independently of the chromosome. 00:02:38.440 --> 00:02:41.200 And they can persist in the bacterial cell 00:02:41.200 --> 00:02:43.390 and be passed on to the daughters 00:02:43.390 --> 00:02:48.520 of this bacterial cell as the bacterial cells divide. 00:02:48.520 --> 00:02:53.200 So if we focus on an individual plasmid, what a plasmid would 00:02:53.200 --> 00:03:01.840 look like, often they have a cassette or a gene that 00:03:01.840 --> 00:03:05.260 confers antibiotic resistance. 00:03:05.260 --> 00:03:09.340 And that's often the reason that these bacteria are harboring 00:03:09.340 --> 00:03:13.510 these plasmids, because it gives them a sort of advantage 00:03:13.510 --> 00:03:16.450 if they're exposed to a certain antibiotic 00:03:16.450 --> 00:03:19.460 from a predator organism. 00:03:19.460 --> 00:03:23.590 So this would confer antibiotic resistance. 00:03:27.820 --> 00:03:30.770 One common example is ampicillin resistance, 00:03:30.770 --> 00:03:35.380 which I'll abbreviate just amp with an R next to it. 00:03:35.380 --> 00:03:40.030 But these plasmids, for them to propagate from bacterial cell 00:03:40.030 --> 00:03:42.100 to bacterial cell, they also need 00:03:42.100 --> 00:03:44.680 to be able to undergo replication. 00:03:44.680 --> 00:03:52.720 So they also have what is known as an origin of replication, 00:03:52.720 --> 00:03:57.880 which is often abbreviated ori, which basically allows 00:03:57.880 --> 00:04:03.850 this plasmid to be replicated such that copies of the plasma 00:04:03.850 --> 00:04:10.390 are passed on to the subsequent generation of bacteria. 00:04:10.390 --> 00:04:13.750 And we can take advantage of this plasmid system 00:04:13.750 --> 00:04:17.450 in bacteria, because we could take, let's say, 00:04:17.450 --> 00:04:20.420 a piece of foreign DNA-- 00:04:20.420 --> 00:04:23.980 and this foreign DNA could be of eukaryotic origin. 00:04:26.500 --> 00:04:29.620 We could take a piece of eukaryotic DNA 00:04:29.620 --> 00:04:31.900 and insert it inside of this plasmid. 00:04:34.900 --> 00:04:40.780 And we can basically use the plasma as a sort of platform 00:04:40.780 --> 00:04:44.260 or a vector to carry the piece of DNA 00:04:44.260 --> 00:04:46.960 that we might be interested in and to use 00:04:46.960 --> 00:04:51.370 the bacteria to replicate that DNA and pass it 00:04:51.370 --> 00:04:53.530 on to its descendants so that you essentially 00:04:53.530 --> 00:04:56.110 have a clone of bacteria, and you 00:04:56.110 --> 00:05:01.150 have a clone of this DNA in a given bacterial population. 00:05:01.150 --> 00:05:04.210 So, again, this would have an origin and maybe 00:05:04.210 --> 00:05:06.550 an ampicillin resistance to it. 00:05:09.170 --> 00:05:11.350 So how would you determine whether or not 00:05:11.350 --> 00:05:15.416 bacteria have a plasmid in it? 00:05:15.416 --> 00:05:17.110 Can you think of an experiment you 00:05:17.110 --> 00:05:22.730 could do to determine whether the bacteria has this plasmid? 00:05:22.730 --> 00:05:23.410 Stephen? 00:05:23.410 --> 00:05:24.868 AUDIENCE: You could add ampicillin, 00:05:24.868 --> 00:05:26.555 and it'll survive with the plasmid. 00:05:26.555 --> 00:05:27.430 ADAM MARTIN: Exactly. 00:05:27.430 --> 00:05:31.360 So what Stephen suggested is that if he 00:05:31.360 --> 00:05:34.510 wanted to know whether or not this bacteria had 00:05:34.510 --> 00:05:38.950 the plasmid in it, he would add ampicillin to the media. 00:05:38.950 --> 00:05:42.190 And if the bacteria doesn't have the plasmid, 00:05:42.190 --> 00:05:43.940 it won't be able to grow. 00:05:43.940 --> 00:05:47.560 But if it has the plasma, it encodes this gene 00:05:47.560 --> 00:05:50.380 that confers ampicillin resistance, 00:05:50.380 --> 00:05:53.530 and it will thus be able to grow. 00:05:53.530 --> 00:05:54.860 So that's exactly right. 00:05:54.860 --> 00:05:59.950 So you're able to select for bacteria with a given plasmid 00:05:59.950 --> 00:06:06.280 by simply growing it on media that contains an antibiotic. 00:06:06.280 --> 00:06:11.410 I now want to go through steps in cloning a piece of DNA. 00:06:11.410 --> 00:06:15.960 And we'll go through it sort of as a series of ordered steps 00:06:15.960 --> 00:06:18.980 so you can see how the process works. 00:06:18.980 --> 00:06:23.155 I'm going to start with a step to cut the DNA. 00:06:25.930 --> 00:06:29.585 After cutting the DNA, we'll then mix pieces together. 00:06:32.440 --> 00:06:34.930 Once we mix the pieces together, we'll 00:06:34.930 --> 00:06:37.060 do something known as a ligation, 00:06:37.060 --> 00:06:41.030 and I'll explain that to you in just a minute. 00:06:41.030 --> 00:06:44.470 And then, finally, we'll end with selecting 00:06:44.470 --> 00:06:51.140 the plasmids that have the piece of foreign DNA that we want. 00:06:51.140 --> 00:06:53.650 And this is known as recombinant DNA, 00:06:53.650 --> 00:06:57.130 because you've recombined a piece of DNA from one 00:06:57.130 --> 00:06:59.890 organism-- it could be a eukaryotic organism, 00:06:59.890 --> 00:07:01.630 like humans-- 00:07:01.630 --> 00:07:05.110 with a piece of DNA that's from a prokaryotic cell, 00:07:05.110 --> 00:07:07.360 a bacterium. 00:07:07.360 --> 00:07:09.940 So we then have some sort of selection process. 00:07:13.540 --> 00:07:17.260 So we're going to go through this step-by-step. 00:07:17.260 --> 00:07:20.180 And we're going to start with cutting DNA, OK? 00:07:23.590 --> 00:07:24.535 So, cutting DNA. 00:07:30.040 --> 00:07:33.070 And it turns out, we've talked about-- 00:07:33.070 --> 00:07:36.130 what type of enzyme do you think would cut DNA? 00:07:42.200 --> 00:07:45.590 Just generally, not as specific as what's up on the slide. 00:07:45.590 --> 00:07:49.520 What type of enzyme would cleave DNA? 00:07:49.520 --> 00:07:54.282 Think about how enzymes are named. 00:07:54.282 --> 00:07:55.240 AUDIENCE: DNase? 00:07:55.240 --> 00:07:57.020 ADAM MARTIN: Yeah, so Stephen suggested 00:07:57.020 --> 00:08:00.590 DNase, which is a really good suggestion, right? 00:08:00.590 --> 00:08:05.090 So an enzyme that will cut DNA would be a DNase. 00:08:05.090 --> 00:08:07.010 Another word for that is a nuclease. 00:08:07.010 --> 00:08:08.950 It's some type of nuclease. 00:08:08.950 --> 00:08:12.140 And the type of nuclease we're going to talk about 00:08:12.140 --> 00:08:16.040 is going to be an endonuclease. 00:08:16.040 --> 00:08:18.200 We talked about exonucleases, which 00:08:18.200 --> 00:08:23.640 chew DNA from the ends in the context of DNA replication. 00:08:23.640 --> 00:08:26.330 But what an endonuclease is, is it's 00:08:26.330 --> 00:08:34.590 a nuclease that's going to recognize a fragment of DNA 00:08:34.590 --> 00:08:36.000 and cleave it in the middle. 00:08:36.000 --> 00:08:37.740 So it doesn't require an end. 00:08:37.740 --> 00:08:41.250 It's going to chop it right in the middle. 00:08:41.250 --> 00:08:45.370 And there's a type of endonuclease, 00:08:45.370 --> 00:08:48.630 and these are called restriction endonucleases. 00:08:48.630 --> 00:08:51.300 They are nucleases that our natively present 00:08:51.300 --> 00:08:55.070 in a lot of different bacteria. 00:08:55.070 --> 00:08:58.370 And these restriction endonucleases essentially 00:08:58.370 --> 00:09:01.010 look through the DNA sequence, and they 00:09:01.010 --> 00:09:06.290 recognize a specific sequence of nucleotides 00:09:06.290 --> 00:09:10.070 and make a cut right at that sequence. 00:09:10.070 --> 00:09:12.870 So I have a few examples to show you here. 00:09:12.870 --> 00:09:17.180 The first is this EcoR1 restriction endonuclease. 00:09:17.180 --> 00:09:24.760 EcoR1's from E. coli, and it recognizes the sequence GAATTC. 00:09:24.760 --> 00:09:27.320 So it recognizes this six-nucleotide . 00:09:27.320 --> 00:09:31.160 Sequence and it cleaves the double-stranded DNA 00:09:31.160 --> 00:09:33.985 on the top strand between the G and the A 00:09:33.985 --> 00:09:37.880 and on the bottom strand between the G and the A, OK? 00:09:37.880 --> 00:09:40.790 And you can see that the two cuts are staggered. 00:09:40.790 --> 00:09:45.770 So when this cut is made, it leaves the DNA with two ends, 00:09:45.770 --> 00:09:51.020 and they're sticky, because there's a five-prime overhang 00:09:51.020 --> 00:09:52.430 at each end. 00:09:52.430 --> 00:09:55.805 So each end has this TTAA sequence. 00:09:58.850 --> 00:10:01.700 And these nucleotide bases can base pair 00:10:01.700 --> 00:10:04.910 with the complementary sequence. 00:10:04.910 --> 00:10:08.030 So this sequence could base pair with a sequence 00:10:08.030 --> 00:10:09.290 that has an end AATT. 00:10:11.900 --> 00:10:15.290 So these two ends that I've generated here 00:10:15.290 --> 00:10:17.210 could stick to each other, or there 00:10:17.210 --> 00:10:21.560 could be other ends that have the TTAA sequence that 00:10:21.560 --> 00:10:24.590 could stick to them. 00:10:24.590 --> 00:10:28.880 So another example is this Kpn1 endonuclease. 00:10:28.880 --> 00:10:31.280 And this is from a different bacterium. 00:10:31.280 --> 00:10:35.000 But again, it cleaves the DNA on the two strands. 00:10:35.000 --> 00:10:38.270 And this time, it cleaves the top strand 00:10:38.270 --> 00:10:40.370 farther down the sequence. 00:10:40.370 --> 00:10:43.440 And that generates what's known as a three-prime overhang. 00:10:43.440 --> 00:10:45.260 But again, you have an overhang. 00:10:45.260 --> 00:10:48.410 So this is what is known as having a sticky end. 00:10:48.410 --> 00:10:50.840 Because again, these nucleotides are 00:10:50.840 --> 00:10:55.430 available to base pair with a complementary sequence. 00:10:55.430 --> 00:10:57.560 The final type of restriction enzyme 00:10:57.560 --> 00:11:00.560 that I'll tell you about is EcoR5, 00:11:00.560 --> 00:11:03.770 which is a different enzyme from E. coli. 00:11:03.770 --> 00:11:06.650 And this generates a break, but this time, it 00:11:06.650 --> 00:11:10.700 cuts at the same position on both DNA strands. 00:11:10.700 --> 00:11:14.390 And that generates an end that's known as a blunt end, 00:11:14.390 --> 00:11:16.010 because there's no overhang, and there 00:11:16.010 --> 00:11:20.270 are no nucleotides that would sort of basically 00:11:20.270 --> 00:11:25.890 recognize a complementary sort of end like the sticky ends do, 00:11:25.890 --> 00:11:27.620 OK? 00:11:27.620 --> 00:11:31.130 So these are several of many, many different types 00:11:31.130 --> 00:11:33.680 of restriction endonucleases that 00:11:33.680 --> 00:11:35.825 are present in a wide range of bacteria. 00:11:39.830 --> 00:11:42.770 So then, now that you have a tool that 00:11:42.770 --> 00:11:46.130 allows you to cut DNA, you could then cut DNA 00:11:46.130 --> 00:11:47.810 from two different sources. 00:11:47.810 --> 00:11:50.300 And I've outlined that here. 00:11:50.300 --> 00:11:56.060 The vector is what the plasmid DNA is commonly referred to. 00:11:56.060 --> 00:11:59.810 So we commonly refer to this prokaryotic part 00:11:59.810 --> 00:12:03.500 of the plasmid, the vector DNA, and the part 00:12:03.500 --> 00:12:07.060 that we're trying to insert that's the foreign DNA, 00:12:07.060 --> 00:12:09.920 the insert. 00:12:09.920 --> 00:12:11.795 That's just kind of the lingo in the field. 00:12:14.600 --> 00:12:17.300 So here, I have a plasmid. 00:12:17.300 --> 00:12:18.440 It looks like a plasmid. 00:12:18.440 --> 00:12:20.190 It has an origin of replication. 00:12:20.190 --> 00:12:22.400 It has ampicillin resistance. 00:12:22.400 --> 00:12:26.270 And it has this EcoR1 site, which just means that this DNA 00:12:26.270 --> 00:12:30.380 sequence has a GAATTC, OK? 00:12:30.380 --> 00:12:33.260 So it's something that will be recognized by this restriction 00:12:33.260 --> 00:12:34.940 endonuclease. 00:12:34.940 --> 00:12:39.200 And then if you cut this enzyme with EcoR1, 00:12:39.200 --> 00:12:41.360 you start with the linear piece, right? 00:12:41.360 --> 00:12:47.060 So I, at 6:00 AM, started engineering this here. 00:12:47.060 --> 00:12:50.570 So let's say I have my plasmid DNA, 00:12:50.570 --> 00:12:52.700 and I cut it at the EcoR1 site. 00:12:56.220 --> 00:12:57.500 Then I cleave it. 00:12:57.500 --> 00:13:01.770 If you cleave a circle, now you have a linear piece of DNA, OK? 00:13:01.770 --> 00:13:04.620 But it has sticky ends, right? 00:13:04.620 --> 00:13:07.260 And these sticky ends-- 00:13:07.260 --> 00:13:11.110 so pretend that this is a foreign piece of DNA. 00:13:11.110 --> 00:13:13.440 This is my eukaryotic DNA. 00:13:13.440 --> 00:13:16.890 Let's pretend it carries the gene elastin. 00:13:16.890 --> 00:13:19.410 And this has ends, too. 00:13:19.410 --> 00:13:23.310 And if they're EcoR1 ends, then they 00:13:23.310 --> 00:13:27.540 will be able to stick to the sticky ends of the plasmid. 00:13:27.540 --> 00:13:29.370 And if you just get one sticking, 00:13:29.370 --> 00:13:32.100 now you have this piece of DNA which 00:13:32.100 --> 00:13:34.950 is two different fragments in tandem. 00:13:34.950 --> 00:13:36.720 But it's going to be moving around 00:13:36.720 --> 00:13:38.850 in space in the cytoplasm. 00:13:38.850 --> 00:13:43.140 And at some point, it might be recognized and stick 00:13:43.140 --> 00:13:45.450 to the other EcoR1 end. 00:13:45.450 --> 00:13:49.860 And then you have now a circular piece of DNA again, 00:13:49.860 --> 00:13:52.440 but now your circular piece of DNA 00:13:52.440 --> 00:13:55.080 has this piece of foreign DNA that's 00:13:55.080 --> 00:13:59.100 present inside the vector, which is the sort of poster tape 00:13:59.100 --> 00:14:00.630 here. 00:14:00.630 --> 00:14:02.240 So I just wanted to show you that. 00:14:02.240 --> 00:14:03.690 So you can kind of-- 00:14:03.690 --> 00:14:05.190 when you're doing molecular biology, 00:14:05.190 --> 00:14:06.960 you have to imagine the sort of end 00:14:06.960 --> 00:14:09.270 stick sticking to each other and how they're 00:14:09.270 --> 00:14:12.870 going to sort of wrap around and connect for the final product. 00:14:16.590 --> 00:14:20.070 OK, so let's say you get DNA, and your DNA could 00:14:20.070 --> 00:14:25.290 be eukaryotic DNA from, let's say, humans or flies 00:14:25.290 --> 00:14:28.680 or whatever your favorite eukaryote is. 00:14:28.680 --> 00:14:32.700 And in the genome of that organism, 00:14:32.700 --> 00:14:35.640 there will be many restriction sites. 00:14:35.640 --> 00:14:38.010 But if you chop it up, you will get 00:14:38.010 --> 00:14:42.420 various fragments that have sticky ends for EcoR1 00:14:42.420 --> 00:14:43.890 on both sides. 00:14:43.890 --> 00:14:47.730 And then if you mixed the vector and the insert together, 00:14:47.730 --> 00:14:51.030 you have some probability of getting that insert 00:14:51.030 --> 00:14:55.860 to be incorporated into the vector. 00:14:55.860 --> 00:14:59.220 And then once this is present and ligated together, 00:14:59.220 --> 00:15:01.140 you can then put this into bacteria, 00:15:01.140 --> 00:15:02.490 and it will be replicated. 00:15:11.910 --> 00:15:16.770 So we focused on cutting, but if you mix these together, 00:15:16.770 --> 00:15:18.990 like I showed you at the tape, you're 00:15:18.990 --> 00:15:21.210 going to have sticky ends that come together 00:15:21.210 --> 00:15:23.040 and stick together. 00:15:23.040 --> 00:15:25.623 And you'll eventually get a situation 00:15:25.623 --> 00:15:26.790 where you have your insert-- 00:15:30.900 --> 00:15:32.910 so you have your insert here that 00:15:32.910 --> 00:15:37.080 might have your gene of interest and your vector DNA here. 00:15:37.080 --> 00:15:39.960 But when these things initially stick together, 00:15:39.960 --> 00:15:44.130 you don't have a single molecule where everything 00:15:44.130 --> 00:15:45.780 is covalently attached, right? 00:15:45.780 --> 00:15:49.890 You just have these base pair interactions between the two 00:15:49.890 --> 00:15:52.470 overhangs as they stick to each other 00:15:52.470 --> 00:15:55.540 through base pair interactions. 00:15:55.540 --> 00:15:58.530 So if we think about what's going on right here, 00:15:58.530 --> 00:16:03.560 you have, initially, if we're thinking about EcoR1, 00:16:03.560 --> 00:16:05.900 a sequence that is-- 00:16:05.900 --> 00:16:09.900 oh, sorry, this should be a C. This 00:16:09.900 --> 00:16:13.680 is the nucleotide sequence, but when it's sticking together, 00:16:13.680 --> 00:16:18.630 the top strand will have a free five-prime phosphate 00:16:18.630 --> 00:16:20.400 on this adenosine, and there will 00:16:20.400 --> 00:16:25.470 be no covalent bond between this adenosine and this guanosine. 00:16:25.470 --> 00:16:27.810 So that's what the top strand would look like. 00:16:27.810 --> 00:16:30.260 The bottom strand would look like this, 00:16:30.260 --> 00:16:35.640 where there are covalent bonds along the DNA backbone. 00:16:35.640 --> 00:16:37.830 Sorry. 00:16:37.830 --> 00:16:41.670 Had a mutation there. 00:16:41.670 --> 00:16:45.880 And this is incorporated in a broader sequence. 00:16:45.880 --> 00:16:49.110 And this bottom strand will have a free five-prime phosphate 00:16:49.110 --> 00:16:53.160 here and a free three-prime hydroxyl here, 00:16:53.160 --> 00:16:55.300 but there's no covalent bond here. 00:16:55.300 --> 00:16:57.240 There's no covalent bond here, right? 00:16:57.240 --> 00:17:01.240 So, at this stage, you don't have a single piece of DNA. 00:17:01.240 --> 00:17:05.130 You have two pieces of DNA that are interacting with each other 00:17:05.130 --> 00:17:07.650 through base pair interactions. 00:17:07.650 --> 00:17:11.290 So, eventually, you want it to be a single piece of DNA. 00:17:11.290 --> 00:17:14.940 And so you have to perform a step that is known as-- 00:17:14.940 --> 00:17:18.460 sorry, my phosphate got in the way. 00:17:18.460 --> 00:17:21.750 But you want to perform what's known as a ligation, where you 00:17:21.750 --> 00:17:24.750 take something that's just sticking together 00:17:24.750 --> 00:17:28.020 through nucleotide base pair interactions 00:17:28.020 --> 00:17:31.050 and you add a type of enzyme, which 00:17:31.050 --> 00:17:34.560 is called DNA ligase, to catalyze 00:17:34.560 --> 00:17:39.630 the formation of covalent bonds here and here. 00:17:39.630 --> 00:17:43.380 So DNA ligase is an enzyme that if you 00:17:43.380 --> 00:17:45.810 have a three five-prime phosphate 00:17:45.810 --> 00:17:48.300 here and a free three-prime hydroxyl, 00:17:48.300 --> 00:17:51.480 it'll catalyze the formation of a phosphodiester 00:17:51.480 --> 00:17:56.460 bond between these two bases and the DNA. 00:17:56.460 --> 00:18:00.525 So this DNA ligase forms a phosphodiester bond. 00:18:08.310 --> 00:18:13.110 And you go from having no bond there to having a bond. 00:18:13.110 --> 00:18:19.140 So then you eventually would get this sequence now, 00:18:19.140 --> 00:18:26.640 where you have covalent bonds between each of the base pairs. 00:18:26.640 --> 00:18:31.070 And what you see here is you've recreated the EcoR1 site. 00:18:31.070 --> 00:18:35.040 So when you get two EcoR1 sticky ends 00:18:35.040 --> 00:18:38.220 sort of recognizing each other and sticking to each other 00:18:38.220 --> 00:18:40.290 and then you ligate them together, 00:18:40.290 --> 00:18:44.700 you recreate that nuclease site so that you can cleave it again 00:18:44.700 --> 00:18:46.200 with the EcoR1 enzyme. 00:18:51.770 --> 00:18:53.450 So now, moving on. 00:18:53.450 --> 00:18:55.680 I'll move on right here. 00:18:55.680 --> 00:19:01.490 So the last step is that once we have pieces of DNA 00:19:01.490 --> 00:19:02.270 with this insert-- 00:19:04.940 --> 00:19:09.270 and let's say we're trying to find a piece of DNA 00:19:09.270 --> 00:19:11.640 from a eukaryotic organism. 00:19:11.640 --> 00:19:13.680 We might start with an animal. 00:19:13.680 --> 00:19:16.590 We have to extract its chromosomal DNA, 00:19:16.590 --> 00:19:19.260 digest it with a restriction enzyme, 00:19:19.260 --> 00:19:22.200 and then we digest the vector with the same restriction 00:19:22.200 --> 00:19:23.190 enzyme. 00:19:23.190 --> 00:19:24.920 And then we're going to make-- 00:19:24.920 --> 00:19:27.810 we're going to randomly insert these pieces of DNA 00:19:27.810 --> 00:19:31.320 into different vectors such that each bacteria who 00:19:31.320 --> 00:19:33.990 gets one of these plasmids will be 00:19:33.990 --> 00:19:36.810 replicating a different piece of DNA that's 00:19:36.810 --> 00:19:39.840 of eukaryotic origin. 00:19:39.840 --> 00:19:42.060 And this is what is known as a DNA library. 00:19:44.680 --> 00:19:46.440 So this is making a DNA library. 00:19:49.680 --> 00:19:52.410 And a DNA library is essentially a collection 00:19:52.410 --> 00:19:57.840 of different pieces of DNA that are from some source, OK? 00:19:57.840 --> 00:20:01.110 But different bacterial clones will be replicating 00:20:01.110 --> 00:20:05.220 a different piece of that DNA. 00:20:05.220 --> 00:20:08.010 So you can see the challenge now is 00:20:08.010 --> 00:20:11.460 to find the needle in the haystack, right? 00:20:11.460 --> 00:20:14.400 You're trying to find that one piece of DNA which is 00:20:14.400 --> 00:20:17.550 the one you're interested in. 00:20:17.550 --> 00:20:20.580 And I'll talk about several strategies 00:20:20.580 --> 00:20:23.730 that you can use to find the piece of DNA 00:20:23.730 --> 00:20:25.770 that you're interested in. 00:20:25.770 --> 00:20:27.210 I'll focus on selection. 00:20:27.210 --> 00:20:29.100 But first, I just want to differentiate 00:20:29.100 --> 00:20:31.500 between two different types of ways you could 00:20:31.500 --> 00:20:33.940 search for a piece of DNA. 00:20:33.940 --> 00:20:35.460 You could do a screen. 00:20:35.460 --> 00:20:38.760 And this is similar to what we talked about on Monday, where 00:20:38.760 --> 00:20:42.420 you look through a whole population of individuals, 00:20:42.420 --> 00:20:44.610 and you look for a given phenotype. 00:20:44.610 --> 00:20:48.360 So in the case of flies, we talked 00:20:48.360 --> 00:20:50.640 about how Morgan's lab was looking 00:20:50.640 --> 00:20:53.190 for differences in eye color. 00:20:53.190 --> 00:20:54.870 And that was a screen, because they 00:20:54.870 --> 00:20:56.820 looked through a ton of normal flies 00:20:56.820 --> 00:20:58.860 to find the one they want. 00:20:58.860 --> 00:21:01.290 Another strategy would be to do what's 00:21:01.290 --> 00:21:06.810 called a selection, where you kill off everything that's not 00:21:06.810 --> 00:21:10.770 what you want by making the organism grow 00:21:10.770 --> 00:21:14.220 in some sort of selective condition. 00:21:14.220 --> 00:21:16.320 And then you only allow the organisms 00:21:16.320 --> 00:21:18.810 to grow that are the ones that you want. 00:21:18.810 --> 00:21:21.860 So this is called a selection. 00:21:21.860 --> 00:21:25.200 And I'm going to illustrate several examples of selections, 00:21:25.200 --> 00:21:28.920 just so that you get an idea of how this works. 00:21:28.920 --> 00:21:34.290 So the first example I'll give is antibiotic resistance. 00:21:40.140 --> 00:21:43.620 And as Stephen so kindly pointed out earlier in the lecture, 00:21:43.620 --> 00:21:46.950 the way we can select for bacteria that have taken up 00:21:46.950 --> 00:21:50.220 this plasmid is to select the bacteria that 00:21:50.220 --> 00:21:56.580 grow in the presence of the antibiotic. 00:21:56.580 --> 00:21:59.580 So let's say you had a population of bacteria 00:21:59.580 --> 00:22:01.200 and let's say this started out being 00:22:01.200 --> 00:22:04.080 sensitive to an antibiotic. 00:22:04.080 --> 00:22:07.680 You could transform them with DNA 00:22:07.680 --> 00:22:09.570 from a strain that's resistant. 00:22:14.670 --> 00:22:18.480 And maybe that resistant strain has a plasmid 00:22:18.480 --> 00:22:23.160 that has a gene that confers antibiotic resistance, in which 00:22:23.160 --> 00:22:27.030 case, if it's taken up by this sensitive bacteria, 00:22:27.030 --> 00:22:34.410 if you then grow it on a plate that has the antibiotic, 00:22:34.410 --> 00:22:37.170 you might get a colony or a clone of cells 00:22:37.170 --> 00:22:39.210 that has taken up the piece of DNA 00:22:39.210 --> 00:22:43.050 that you're interested in-- in this case, the piece of DNA 00:22:43.050 --> 00:22:47.070 that confers antibiotic resistance. 00:22:47.070 --> 00:22:49.180 Everyone see how that works? 00:22:49.180 --> 00:22:51.390 So you're selecting only the cells 00:22:51.390 --> 00:22:54.910 to grow here that have taken up this antibiotic resistance 00:22:54.910 --> 00:22:55.410 gene. 00:22:58.890 --> 00:23:02.430 I'm going to use another example now from yeast, 00:23:02.430 --> 00:23:04.840 and it involves functional complementation. 00:23:11.380 --> 00:23:14.490 And I'm going to start with something 00:23:14.490 --> 00:23:18.240 that involves the biosynthesis of an essential amino acid. 00:23:18.240 --> 00:23:21.360 And then I'm going to go to a more interesting case, which 00:23:21.360 --> 00:23:25.050 is a case that involves an experiment that involved 00:23:25.050 --> 00:23:28.410 the identification of the master regulator of cell division 00:23:28.410 --> 00:23:29.790 in humans. 00:23:29.790 --> 00:23:33.890 But we'll start with just amino acid biosynthesis. 00:23:33.890 --> 00:23:39.090 And there are mutants of yeast known as auxotrophs. 00:23:39.090 --> 00:23:44.820 And these are mutant yeast, or mutant microorganisms, 00:23:44.820 --> 00:23:48.360 that fail to produce an essential nutrient. 00:23:48.360 --> 00:23:53.820 So an auxotroph is a mutant that fails to make 00:23:53.820 --> 00:23:59.050 a nutrient that's essential. 00:23:59.050 --> 00:24:02.940 So fails to make a nutrient. 00:24:02.940 --> 00:24:09.630 And the opposite of an auxotroph is called a prototroph. 00:24:09.630 --> 00:24:18.150 A prototroph is essentially a normal-functioning 00:24:18.150 --> 00:24:20.760 microorganism that's able to produce 00:24:20.760 --> 00:24:24.270 all of the essential nutrients that it needs in order 00:24:24.270 --> 00:24:26.550 to grow and survive, OK? 00:24:26.550 --> 00:24:29.160 So this produces all nutrients. 00:24:36.830 --> 00:24:41.340 And so let's say you had an auxotroph for leucine. 00:24:41.340 --> 00:24:45.990 So you had a strain that if you didn't provide leucine to it, 00:24:45.990 --> 00:24:48.690 it would fail to grow. 00:24:48.690 --> 00:24:52.080 So we'll start with a leucine auxotroph. 00:24:54.840 --> 00:24:57.120 And let's say you want to identify 00:24:57.120 --> 00:25:01.170 the gene that's defective in this leucine auxotroph. 00:25:01.170 --> 00:25:06.000 You'd perform a similar strategy to this one, where 00:25:06.000 --> 00:25:10.620 you'd have your auxotroph that you're transforming, 00:25:10.620 --> 00:25:13.260 so your auxotroph down here. 00:25:13.260 --> 00:25:16.740 And you would transform that strain with DNA 00:25:16.740 --> 00:25:18.270 from what organism? 00:25:21.530 --> 00:25:24.590 If you're trying to identify the functional gene, 00:25:24.590 --> 00:25:27.230 what organism would you use to produce 00:25:27.230 --> 00:25:30.654 the DNA you're going to transform into that organism? 00:25:30.654 --> 00:25:31.590 AUDIENCE: Prototroph? 00:25:31.590 --> 00:25:33.440 ADAM MARTIN: Javier's exactly right. 00:25:33.440 --> 00:25:35.390 You'd use the prototroph, right? 00:25:35.390 --> 00:25:40.470 So in this case, you would use DNA from the prototroph, 00:25:40.470 --> 00:25:44.120 because the prototroph has a functional copy of that gene. 00:25:44.120 --> 00:25:46.220 You know it does, because it's able to grow 00:25:46.220 --> 00:25:48.680 without adding leucine. 00:25:48.680 --> 00:25:54.290 And then you could take the auxotroph mutants that's 00:25:54.290 --> 00:25:57.260 transformed with DNA from the prototroph, 00:25:57.260 --> 00:26:00.560 and you played it on media. 00:26:00.560 --> 00:26:04.460 And what should be a property of the media? 00:26:04.460 --> 00:26:06.700 Should leucine be present or absent? 00:26:06.700 --> 00:26:07.272 Carmen? 00:26:07.272 --> 00:26:07.980 AUDIENCE: Absent. 00:26:07.980 --> 00:26:11.120 ADAM MARTIN: It should be absent, exactly right. 00:26:11.120 --> 00:26:13.370 So you'd look on plates that lack 00:26:13.370 --> 00:26:16.010 leucine or minus for leucine. 00:26:16.010 --> 00:26:19.160 And you'd select for colonies that now are all of a sudden 00:26:19.160 --> 00:26:22.040 able to grow leucine. 00:26:22.040 --> 00:26:27.220 So you've restored the function of leucine biosynthesis 00:26:27.220 --> 00:26:30.860 in this clone, and you've made it into a prototroph again. 00:26:34.870 --> 00:26:38.540 OK, this is what's known as functional complementation, 00:26:38.540 --> 00:26:40.580 because you're taking a cell that 00:26:40.580 --> 00:26:45.610 is defective in some function, and you're complementing it. 00:26:45.610 --> 00:26:51.470 You're complementing or rescuing the phenotype, OK? 00:26:51.470 --> 00:26:54.740 Now, even as a former yeast geneticist, 00:26:54.740 --> 00:26:59.060 I don't find amino acid biosynthesis and functional 00:26:59.060 --> 00:27:03.300 complementation in the context of leucine all that exciting. 00:27:03.300 --> 00:27:06.650 So I want to present one last example that 00:27:06.650 --> 00:27:13.220 involves an experiment that is going to involve the yeast cell 00:27:13.220 --> 00:27:13.940 cycle mutants. 00:27:17.360 --> 00:27:20.720 And I'm going to tell you about the experiment that 00:27:20.720 --> 00:27:26.210 led to the cloning of the master regulator of cell division 00:27:26.210 --> 00:27:27.830 in humans. 00:27:27.830 --> 00:27:33.080 And it involves a yeast mutant, and specifically, a yeast cell 00:27:33.080 --> 00:27:35.390 cycle mutant. 00:27:35.390 --> 00:27:37.940 And these yeast cell cycle mutants 00:27:37.940 --> 00:27:41.420 are what are known as conditional mutants. 00:27:41.420 --> 00:27:44.810 They are isolated as conditional mutants, 00:27:44.810 --> 00:27:47.510 meaning that these mutants are able to grow 00:27:47.510 --> 00:27:50.660 under certain conditions, but not others. 00:27:50.660 --> 00:27:55.040 And specifically, the condition they used is temperature, 00:27:55.040 --> 00:27:57.470 so they're temperature-sensitive mutants. 00:27:57.470 --> 00:28:06.020 The yeast cells can grow at 25 degrees, but not at 37 degrees. 00:28:06.020 --> 00:28:09.110 So this is known as a temperature-sensitive mutant, 00:28:09.110 --> 00:28:12.200 where you can propagate the mutant at one temperature, 00:28:12.200 --> 00:28:14.540 but then you can see if you raise the temperature, 00:28:14.540 --> 00:28:16.730 then it stops growing. 00:28:16.730 --> 00:28:18.530 And so you can see the mutant phenotype, 00:28:18.530 --> 00:28:21.530 because normal wild type functional 00:28:21.530 --> 00:28:24.170 yeast can grow at both temperatures. 00:28:24.170 --> 00:28:28.190 So this is a special type of mutant. 00:28:28.190 --> 00:28:30.440 And I'm going to tell you about an experiment done 00:28:30.440 --> 00:28:36.890 by Paul Nurse, who's an excellent yeast geneticist. 00:28:36.890 --> 00:28:41.840 And what he did was he used these yeast cell cycle mutants 00:28:41.840 --> 00:28:45.860 to identify the human gene for what's 00:28:45.860 --> 00:28:57.860 now known as cyclin-dependent kinase, or CDK for short. 00:28:57.860 --> 00:29:01.010 This is the master regulator of cell division 00:29:01.010 --> 00:29:05.810 in organisms ranging from yeast all the way up to humans, OK? 00:29:05.810 --> 00:29:11.210 But he used yeast as a model system to identify this gene. 00:29:11.210 --> 00:29:14.600 And the process was he took yeast cells-- 00:29:14.600 --> 00:29:16.820 and Paul Nurse worked on fission yeast cells, 00:29:16.820 --> 00:29:18.980 which are rod-shaped cells. 00:29:18.980 --> 00:29:20.855 And he identified yeast mutants. 00:29:24.590 --> 00:29:25.370 Yeast mutants. 00:29:25.370 --> 00:29:30.530 And he had a mutant in the CDK gene of yeast. 00:29:30.530 --> 00:29:32.700 He didn't really know it at the time. 00:29:32.700 --> 00:29:36.710 But the yeast CDK mutant-- 00:29:36.710 --> 00:29:40.010 what he knew was that this mutant was critically involved 00:29:40.010 --> 00:29:42.770 in the cell cycle in numerous types of yeast. 00:29:42.770 --> 00:29:44.960 So he knew this is an important gene. 00:29:44.960 --> 00:29:47.780 And what he wanted to do was to identify 00:29:47.780 --> 00:29:50.030 if humans had an equivalent gene that 00:29:50.030 --> 00:29:53.780 could function in the same way. 00:29:53.780 --> 00:29:57.530 So if you just have this CDK mutant and do nothing to it, 00:29:57.530 --> 00:30:01.160 it will not grow at 37 degrees, OK? 00:30:01.160 --> 00:30:05.990 But what he did was to take a DNA library-- 00:30:05.990 --> 00:30:07.670 similar to what I showed you before, 00:30:07.670 --> 00:30:11.900 where you just chop up DNA from an organism. 00:30:11.900 --> 00:30:15.090 In this case, he's using a human DNA library. 00:30:15.090 --> 00:30:17.060 And he used a particular type of library, 00:30:17.060 --> 00:30:19.135 but I'm going to skip over that for now 00:30:19.135 --> 00:30:21.040 and come back to it later. 00:30:21.040 --> 00:30:25.170 So he used a human DNA library. 00:30:25.170 --> 00:30:26.910 That's just a collection of pieces 00:30:26.910 --> 00:30:30.210 of DNA from a human source, OK? 00:30:30.210 --> 00:30:33.780 So he's taking human DNA, putting it 00:30:33.780 --> 00:30:36.930 into a yeast plasmid, and transforming yeast 00:30:36.930 --> 00:30:38.790 with that human DNA. 00:30:38.790 --> 00:30:41.370 And he's looking for a piece of DNA 00:30:41.370 --> 00:30:45.330 that's able to complement the CDK mutant, 00:30:45.330 --> 00:30:47.220 meaning the yeast cells would then 00:30:47.220 --> 00:30:50.250 be able to grow at the non-permissive temperature 00:30:50.250 --> 00:30:53.070 of 37 degrees. 00:30:53.070 --> 00:30:57.390 So he then looks for, on a plate, colonies of yeast 00:30:57.390 --> 00:31:00.030 that are growing at the non-permissive temperature 00:31:00.030 --> 00:31:01.810 of 37 degrees. 00:31:01.810 --> 00:31:03.930 So if you didn't do anything with this mutant, 00:31:03.930 --> 00:31:07.020 if you didn't transform in the DNA, nothing would grow. 00:31:07.020 --> 00:31:10.230 But he identified pieces of human DNA 00:31:10.230 --> 00:31:16.310 that rescued the phenotype of this mutant, OK? 00:31:16.310 --> 00:31:24.930 And so these are yeast that have the human gene for CDK, 00:31:24.930 --> 00:31:27.690 and they now grow. 00:31:27.690 --> 00:31:31.050 And this is a functional complementation experiment, 00:31:31.050 --> 00:31:34.750 because you're rescuing the growth of this yeast 00:31:34.750 --> 00:31:38.850 now not with a yeast gene, but with a human gene. 00:31:38.850 --> 00:31:43.770 And this human CDK gene is so conserved across eukaryotes 00:31:43.770 --> 00:31:47.010 that it's able to still function in a yeast cell, which 00:31:47.010 --> 00:31:48.940 is pretty amazing. 00:31:48.940 --> 00:31:51.996 So this just outlines the experiment here. 00:31:51.996 --> 00:31:54.690 At 25 degrees, these yeast mutants 00:31:54.690 --> 00:31:57.750 can grow and form colonies. 00:31:57.750 --> 00:32:00.900 And at that temperature, you can transform the yeast 00:32:00.900 --> 00:32:04.650 with different pieces of human DNA. 00:32:04.650 --> 00:32:07.530 Most of the human DNA is not going to be what you want. 00:32:07.530 --> 00:32:09.660 You're looking for the needle in the haystack. 00:32:09.660 --> 00:32:12.810 So most of these are not going to grow at 37 degree. 00:32:12.810 --> 00:32:16.950 But you're looking for this guy here that gets the human CDK, 00:32:16.950 --> 00:32:21.750 and that restores growth to this mutant strain. 00:32:21.750 --> 00:32:25.320 So voila, you get a colony of cells that are growing. 00:32:25.320 --> 00:32:28.020 And boom, Paul Nurse wins a Nobel Prize 00:32:28.020 --> 00:32:31.440 and the rest of the yeast field, as well, or a number of people 00:32:31.440 --> 00:32:33.990 who are working on cell cycle mutants. 00:32:33.990 --> 00:32:35.820 This is one of the experiments that 00:32:35.820 --> 00:32:41.370 led to the 2001 Nobel Prize for a bunch of yeast cell cycle 00:32:41.370 --> 00:32:44.360 geneticists. 00:32:44.360 --> 00:32:46.710 All right, so I've told you about how to find 00:32:46.710 --> 00:32:47.835 the needle in the haystack. 00:32:50.730 --> 00:32:55.500 And this was more common when we didn't know the genome sequence 00:32:55.500 --> 00:32:56.760 of an organism. 00:32:56.760 --> 00:33:00.420 But now I want to tell you how knowing the genome sequence 00:33:00.420 --> 00:33:04.620 of an organism would allow you to replicate and amplify 00:33:04.620 --> 00:33:11.280 a piece of DNA in vitro. 00:33:11.280 --> 00:33:15.630 So I'm going to tell you about an approach known as Polymerase 00:33:15.630 --> 00:33:19.140 Chain Reaction, or PCR. 00:33:19.140 --> 00:33:24.600 And what PCR is, is it's an in vitro method. 00:33:24.600 --> 00:33:31.920 So it's an in vitro approach to essentially amplify DNA. 00:33:36.180 --> 00:33:38.880 And so let's say you have a piece of DNA-- 00:33:38.880 --> 00:33:41.400 it could be a piece of DNA in the genome-- 00:33:41.400 --> 00:33:43.230 and you know the sequence of this DNA. 00:33:46.180 --> 00:33:51.300 And it has base pairs between the two strands. 00:33:51.300 --> 00:33:53.910 So, normally, for DNA replication to occur, 00:33:53.910 --> 00:33:56.387 what do you need? 00:33:56.387 --> 00:33:57.470 What needs to happen here? 00:34:01.340 --> 00:34:04.260 Can a polymerase get in now? 00:34:04.260 --> 00:34:04.760 No? 00:34:04.760 --> 00:34:05.750 Why not? 00:34:05.750 --> 00:34:07.156 Miles? 00:34:07.156 --> 00:34:09.621 AUDIENCE: The DNA's going to-- 00:34:09.621 --> 00:34:12.579 because they'll try and [INAUDIBLE] away [INAUDIBLE] 00:34:12.579 --> 00:34:14.550 from each other, so you have to [INAUDIBLE].. 00:34:14.550 --> 00:34:16.550 ADAM MARTIN: Yeah, you have to unwind it, right? 00:34:16.550 --> 00:34:20.239 So you have to denature it first. 00:34:20.239 --> 00:34:23.510 So if you do nature it, now you have two single-stranded pieces 00:34:23.510 --> 00:34:26.510 of DNA, right? 00:34:26.510 --> 00:34:31.570 Now what would a polymerase need to replicate that? 00:34:31.570 --> 00:34:32.290 Yeah, Jeremy? 00:34:32.290 --> 00:34:33.040 AUDIENCE: A prime. 00:34:33.040 --> 00:34:33.957 ADAM MARTIN: A primer. 00:34:33.957 --> 00:34:35.040 Exactly, right? 00:34:35.040 --> 00:34:37.170 And if you know the sequence, you 00:34:37.170 --> 00:34:40.590 can have a company synthesize a primer that's 00:34:40.590 --> 00:34:44.520 the exact sequence here and base pairs here. 00:34:44.520 --> 00:34:46.650 And I'll just draw the five-prime end of the primer 00:34:46.650 --> 00:34:47.699 right there. 00:34:47.699 --> 00:34:51.840 And now this primer has a free three-prime hydroxyl here. 00:34:51.840 --> 00:34:53.639 And if you added a polymerase, it 00:34:53.639 --> 00:34:56.820 would synthesize this bottom strand here. 00:34:56.820 --> 00:34:58.515 So this is known as the forward primer. 00:35:01.720 --> 00:35:03.640 And on the other strand, you can design 00:35:03.640 --> 00:35:08.150 a primer that's complementary to these bases here. 00:35:08.150 --> 00:35:10.780 Again, the five-primer end is out. 00:35:10.780 --> 00:35:13.600 This would be known as the reverse primer. 00:35:13.600 --> 00:35:16.480 And then you could have the DNA polymerase 00:35:16.480 --> 00:35:18.670 synthesize the opposite strand. 00:35:21.640 --> 00:35:24.460 All right, so the step here will be to first denature. 00:35:24.460 --> 00:35:28.310 So the first step would be to melt or denature 00:35:28.310 --> 00:35:30.355 the DNA, double-stranded DNA. 00:35:32.920 --> 00:35:35.630 So you denature the double-stranded DNA. 00:35:35.630 --> 00:35:40.870 This is commonly done above 90 degrees Celsius. 00:35:40.870 --> 00:35:42.460 And then the next step is once you 00:35:42.460 --> 00:35:45.400 have these single-stranded pieces of DNA, 00:35:45.400 --> 00:35:49.540 you can act you can have a primer present that anneals 00:35:49.540 --> 00:35:51.730 to the opposite strands. 00:35:51.730 --> 00:35:53.850 So you can have primer annealing. 00:35:59.020 --> 00:36:03.880 And this is commonly done between 50 and 60 degrees 00:36:03.880 --> 00:36:04.670 Celsius. 00:36:04.670 --> 00:36:08.290 You have to cool it down so that the primer can now base pair, 00:36:08.290 --> 00:36:10.600 such that not everything is denatured. 00:36:10.600 --> 00:36:12.670 So you have to cool it down for these primers 00:36:12.670 --> 00:36:16.030 to recognize their cognate sequence and base pair with it. 00:36:19.670 --> 00:36:21.970 And then once you have the primer annealed 00:36:21.970 --> 00:36:26.530 to the template, then you can add DNA polymerase 00:36:26.530 --> 00:36:29.080 to synthesize a new strand. 00:36:29.080 --> 00:36:35.440 So DNA polymerize for new strand synthesis. 00:36:35.440 --> 00:36:41.300 And this is commonly done at around 70 degrees Celsius. 00:36:41.300 --> 00:36:45.240 And then you can repeat this process over and over again. 00:36:45.240 --> 00:36:48.160 And at each step, you're going to double the amount of DNA 00:36:48.160 --> 00:36:52.420 that you have between these two primers. 00:36:52.420 --> 00:36:55.540 So let me just-- this is just a figure illustrating this. 00:36:55.540 --> 00:36:57.940 It's on the handout and online. 00:36:57.940 --> 00:37:01.540 Basically, you have your original double-stranded piece 00:37:01.540 --> 00:37:02.870 of DNA. 00:37:02.870 --> 00:37:05.860 You denature it and allow the primers to anneal. 00:37:05.860 --> 00:37:08.320 New strand synthesis. 00:37:08.320 --> 00:37:13.260 Then you take these new pieces of double-stranded DNA, 00:37:13.260 --> 00:37:14.890 denature them. 00:37:14.890 --> 00:37:17.950 The primers anneal to those new strands, 00:37:17.950 --> 00:37:19.540 and now you get new strands. 00:37:19.540 --> 00:37:22.990 And you just keep doing this cycle over and over again, 00:37:22.990 --> 00:37:26.620 and you essentially amplify the piece of DNA 00:37:26.620 --> 00:37:30.010 that's between the two primer sequences. 00:37:30.010 --> 00:37:31.710 So this is often used in forensics, 00:37:31.710 --> 00:37:34.690 because you can have very little DNA, 00:37:34.690 --> 00:37:37.930 and just by adding primers, you can really 00:37:37.930 --> 00:37:41.080 amplify the number of pieces of DNA 00:37:41.080 --> 00:37:43.360 you have between these two fragments. 00:37:43.360 --> 00:37:47.980 So you go from having very little DNA to a lot of DNA. 00:37:47.980 --> 00:37:51.460 OK, any questions about PCR? 00:37:51.460 --> 00:37:55.620 I'm going to move on to something that-- 00:37:55.620 --> 00:37:56.120 all right. 00:37:59.030 --> 00:38:02.190 I've really been focused on discovery up to this point. 00:38:02.190 --> 00:38:05.270 But I know that a number of you are engineers, 00:38:05.270 --> 00:38:09.470 and you probably want to engineer something. 00:38:09.470 --> 00:38:10.940 And so I've had to-- 00:38:14.030 --> 00:38:16.010 I'm going to tell you about a field that 00:38:16.010 --> 00:38:19.040 is moving so rapidly, I'm going to probably have 00:38:19.040 --> 00:38:22.380 to totally revamp my lecture for next year, OK? 00:38:22.380 --> 00:38:26.120 And I'm going to tell you about genome editing. 00:38:26.120 --> 00:38:31.250 So the last part of this story, genome or DNA editing. 00:38:31.250 --> 00:38:35.420 And I'm going to tell you about a specific type of system 00:38:35.420 --> 00:38:39.530 called CRISPR-Cas9, which has been in the news a lot, 00:38:39.530 --> 00:38:42.170 and there's a lot of excitement about this approach 00:38:42.170 --> 00:38:45.020 in the context of editing the human genome 00:38:45.020 --> 00:38:47.870 and possibly curing genetic diseases. 00:38:47.870 --> 00:38:50.690 Who here has heard of CRISPR-Cas9? 00:38:50.690 --> 00:38:53.040 OK, good. 00:38:53.040 --> 00:38:53.540 That's good. 00:38:53.540 --> 00:38:54.875 Our media is doing its job. 00:38:57.620 --> 00:39:01.080 So who knows what it is? 00:39:01.080 --> 00:39:03.120 OK, some of us know what it is. 00:39:03.120 --> 00:39:06.600 I just want to just give you a very superficial overview 00:39:06.600 --> 00:39:08.735 of what it is and why it's important. 00:39:08.735 --> 00:39:10.110 And I'm going to keep coming back 00:39:10.110 --> 00:39:12.270 to it during the course of the semester, 00:39:12.270 --> 00:39:15.710 because I think it raises a lot of ethical questions, 00:39:15.710 --> 00:39:18.120 and especially in the context of stem cells. 00:39:18.120 --> 00:39:20.160 I need you to know the foundation before we 00:39:20.160 --> 00:39:24.090 get into the really good stuff. 00:39:24.090 --> 00:39:26.460 So, let's see. 00:39:26.460 --> 00:39:29.220 So we're going to engineer something. 00:39:29.220 --> 00:39:32.710 So we're going to talk about repairing DNA. 00:39:32.710 --> 00:39:34.860 And if we want to edit the genome, 00:39:34.860 --> 00:39:37.410 the way this is most often done is 00:39:37.410 --> 00:39:41.040 by making a double-stranded break, OK? 00:39:41.040 --> 00:39:45.090 So if you make a double-stranded break in a piece of DNA, 00:39:45.090 --> 00:39:47.770 it can be repaired one of two ways. 00:39:47.770 --> 00:39:49.770 One is by non-homologous end joining, 00:39:49.770 --> 00:39:52.200 where the two pieces of DNA are basically just shoved 00:39:52.200 --> 00:39:53.730 back together again. 00:39:53.730 --> 00:39:56.370 And this results, often, in mutations. 00:39:56.370 --> 00:39:58.117 So if you're trying to fix something, 00:39:58.117 --> 00:39:59.700 unless you're just trying to break it, 00:39:59.700 --> 00:40:01.770 that's probably not what you want. 00:40:01.770 --> 00:40:04.380 But an alternative approach to DNA 00:40:04.380 --> 00:40:07.200 repair that organisms have is something called 00:40:07.200 --> 00:40:09.800 homology-directed repair. 00:40:09.800 --> 00:40:12.660 In this case, you can break a piece of DNA 00:40:12.660 --> 00:40:16.170 and add a piece of DNA that has a different sequence, 00:40:16.170 --> 00:40:20.350 but with homology near where the double-stranded break is. 00:40:20.350 --> 00:40:24.360 And in that case, you can replace the original sequence 00:40:24.360 --> 00:40:27.690 with what you provide as donor DNA. 00:40:27.690 --> 00:40:30.150 So it gives you an ability to essentially 00:40:30.150 --> 00:40:33.630 change the DNA sequence at a given locus 00:40:33.630 --> 00:40:39.360 if you're able to cleave the DNA at a specific locus. 00:40:39.360 --> 00:40:42.240 So, first, I want to start with just a thought 00:40:42.240 --> 00:40:43.470 experiment, right? 00:40:43.470 --> 00:40:48.030 You're all thinking, OK, we need to cleave double-stranded DNA. 00:40:48.030 --> 00:40:50.820 And boy, I just gave you a perfect tool for that. 00:40:50.820 --> 00:40:54.030 I gave you all these restriction endonucleases, right? 00:40:54.030 --> 00:40:55.620 So what's the problem with those? 00:40:55.620 --> 00:40:58.260 Well, let's think about the human genome. 00:40:58.260 --> 00:41:02.490 The human genome is 3 billion base pairs. 00:41:05.580 --> 00:41:08.230 And an EcoR1 site looks like this-- 00:41:08.230 --> 00:41:08.730 GAATTC. 00:41:11.880 --> 00:41:15.000 So it's six nucleotides long. 00:41:15.000 --> 00:41:17.790 And if you think of just a random sequence 00:41:17.790 --> 00:41:21.780 of 3 billion base pairs, you would 00:41:21.780 --> 00:41:25.890 get this sequence randomly one out of every four 00:41:25.890 --> 00:41:28.350 to the sixth times. 00:41:28.350 --> 00:41:35.010 So that's going to be one every 4,096 times. 00:41:35.010 --> 00:41:39.930 So if you get this in random DNA 1 every roughly 4,000 times, 00:41:39.930 --> 00:41:42.410 if you use it to cleave the human genome, 00:41:42.410 --> 00:41:44.760 it's going to cleave hundreds of thousands 00:41:44.760 --> 00:41:47.460 of places in the human genome. 00:41:47.460 --> 00:41:50.280 So we need much more specificity if we 00:41:50.280 --> 00:41:52.080 want to select, let's say, a given 00:41:52.080 --> 00:41:55.800 gene that has a disease-causing allele and try to fix it. 00:41:55.800 --> 00:41:58.140 Because if we use a restriction endonuclease, 00:41:58.140 --> 00:42:02.650 we just chop up the whole genome, and that would be bad. 00:42:02.650 --> 00:42:05.490 So specificity is the name of the game here. 00:42:05.490 --> 00:42:09.030 This is not specific, and we need 00:42:09.030 --> 00:42:11.790 a tool that's more specific. 00:42:11.790 --> 00:42:15.150 And that tool is going to be CRISPR-Cas9. 00:42:15.150 --> 00:42:18.870 And what CRISPR-Cas9 is, is it's essentially 00:42:18.870 --> 00:42:23.060 an RNA-guided endonuclease. 00:42:23.060 --> 00:42:29.070 So it's RNA guided, and it's an endonuclease. 00:42:29.070 --> 00:42:32.070 Restriction enzymes, right, they have nothing to do with RNA. 00:42:32.070 --> 00:42:36.870 They don't use RNA to recognize the nucleotide sequence. 00:42:36.870 --> 00:42:38.490 It's just a protein, and the protein 00:42:38.490 --> 00:42:41.070 recognizes the nucleotide sequence. 00:42:41.070 --> 00:42:48.015 In CRISPR-Cas9, you have an endonuclease, which is Cas9. 00:42:48.015 --> 00:42:51.500 Let's bump this up. 00:42:51.500 --> 00:42:53.720 So the endonuclease is the-- 00:42:53.720 --> 00:42:55.700 the Cas9 is the protein. 00:42:55.700 --> 00:42:57.110 That's the endonuclease. 00:43:01.400 --> 00:43:06.440 But its selection of a target depends on an RNA molecule 00:43:06.440 --> 00:43:08.600 that it's bound to, OK? 00:43:08.600 --> 00:43:14.000 So the specificity comes, at least in part, 00:43:14.000 --> 00:43:20.270 from what's known as a guide RNA, or single guide RNA. 00:43:20.270 --> 00:43:24.930 That's what's most often used in genome editing. 00:43:24.930 --> 00:43:29.180 So this guide RNA basically allows this enzyme 00:43:29.180 --> 00:43:32.030 to find a specific sequence. 00:43:32.030 --> 00:43:34.730 And the guide RNA is 20 nucleotides, 00:43:34.730 --> 00:43:38.420 or looks for homology for a 20-nucleotide base pair 00:43:38.420 --> 00:43:39.620 sequence. 00:43:39.620 --> 00:43:43.040 So you can see, already, we're doing way better than the six 00:43:43.040 --> 00:43:45.140 base pair recognition motif. 00:43:45.140 --> 00:43:46.910 We have 20 nucleotides. 00:43:46.910 --> 00:43:49.040 And there are other components of the system 00:43:49.040 --> 00:43:53.000 which increase the specificity. 00:43:53.000 --> 00:43:55.220 Then you have your Cas9 in blue, which 00:43:55.220 --> 00:43:59.350 is the endonuclease, your RNA, the guide RNA, in black, 00:43:59.350 --> 00:44:01.640 and the template here is in gray. 00:44:01.640 --> 00:44:03.680 And what you see is this RNA sort 00:44:03.680 --> 00:44:08.570 of exhibiting complementarity to this target sequence. 00:44:08.570 --> 00:44:12.260 And only if there's complementarity between the RNA 00:44:12.260 --> 00:44:15.140 and the target will this endonuclease 00:44:15.140 --> 00:44:18.800 get activated and cleave at this site. 00:44:18.800 --> 00:44:22.220 So the RNA is sort of serving like a guide dog 00:44:22.220 --> 00:44:27.345 for this endonuclease to guide it to a certain location 00:44:27.345 --> 00:44:27.845 to cleave. 00:44:34.370 --> 00:44:37.640 So the idea, then, is if you want to edit the genome-- 00:44:37.640 --> 00:44:40.760 and why people are so excited about this 00:44:40.760 --> 00:44:44.240 these days is you now have a system that 00:44:44.240 --> 00:44:48.710 might allow you to generate a double-stranded break in one 00:44:48.710 --> 00:44:51.230 specific place in the genome. 00:44:51.230 --> 00:44:55.880 And if you can do that, then if you provide donor DNA that 00:44:55.880 --> 00:44:59.060 maybe has a different sequence-- if you consider a disease 00:44:59.060 --> 00:44:59.870 allele, right? 00:44:59.870 --> 00:45:03.440 Let's say you know there's a gene that when 00:45:03.440 --> 00:45:08.390 there's a certain allele causes an inherited form of a disease. 00:45:08.390 --> 00:45:13.340 You could then take donor DNA from an unaffected individual 00:45:13.340 --> 00:45:16.730 and take cells from the affected individual 00:45:16.730 --> 00:45:19.790 and cut the locus that's problematic 00:45:19.790 --> 00:45:25.220 and get a repair of the defective allele using 00:45:25.220 --> 00:45:27.470 a normal allele of the gene. 00:45:27.470 --> 00:45:29.390 And that would essentially rescue 00:45:29.390 --> 00:45:32.030 the function of that gene if it were then reintroduced 00:45:32.030 --> 00:45:33.655 into the patient, OK? 00:45:33.655 --> 00:45:38.270 Do you see sort of roughly how this works? 00:45:38.270 --> 00:45:41.960 So this is a very sort of broad and general sort 00:45:41.960 --> 00:45:44.810 of conceptual framework for how this might happen. 00:45:44.810 --> 00:45:47.900 Let's say you have an individual with a blood disorder-- 00:45:47.900 --> 00:45:51.350 let's say sickle cell anemia or beta thalassemia. 00:45:51.350 --> 00:45:53.700 Those are inherited blood disorders, 00:45:53.700 --> 00:45:55.730 which lead to anemia. 00:45:55.730 --> 00:45:59.600 You could remove cells, and what might be the best 00:45:59.600 --> 00:46:02.840 are the stem cells from a patient. 00:46:02.840 --> 00:46:05.420 And you could then take those stem cells and use 00:46:05.420 --> 00:46:09.410 CRISPR-Cas9 in vitro in cell culture 00:46:09.410 --> 00:46:14.420 to edit that individual's cells to repair the genetic defect. 00:46:14.420 --> 00:46:16.700 And you could then reintroduce those 00:46:16.700 --> 00:46:19.910 to the patient, where if they're stem cells, 00:46:19.910 --> 00:46:22.520 they'd reoccupy the stem cell niche 00:46:22.520 --> 00:46:24.530 and produce functional blood cells that 00:46:24.530 --> 00:46:27.810 would then essentially cure the individual of the disease. 00:46:27.810 --> 00:46:29.330 This is how scientists are thinking 00:46:29.330 --> 00:46:32.380 about the use of the system nowadays. 00:46:32.380 --> 00:46:35.090 And this hasn't really been successful yet, 00:46:35.090 --> 00:46:37.800 but there are several clinical trials that are currently 00:46:37.800 --> 00:46:41.990 underway, where people are trying 00:46:41.990 --> 00:46:47.390 to show that this can be used to treat human genetic diseases. 00:46:47.390 --> 00:46:50.360 So in the next year, you are going to hear more about this, 00:46:50.360 --> 00:46:55.040 almost undoubtedly, as we start to hear the results of some 00:46:55.040 --> 00:46:56.000 of these patients. 00:46:56.000 --> 00:46:58.290 There are concerns about this, as well. 00:46:58.290 --> 00:47:00.500 I don't want to overblow it. 00:47:00.500 --> 00:47:01.730 There are certainly concerns. 00:47:01.730 --> 00:47:03.560 We don't know this is going to work. 00:47:03.560 --> 00:47:06.020 I mean, people have been talking about this type of stuff 00:47:06.020 --> 00:47:08.570 since I was a student 20 years ago. 00:47:08.570 --> 00:47:11.900 But I feel like we're getting-- we're much more advanced now, 00:47:11.900 --> 00:47:13.520 and the tools are more advanced. 00:47:13.520 --> 00:47:17.300 And so I feel like we're kind of getting to the point where 00:47:17.300 --> 00:47:19.910 there's a much greater chance that this will be successful 00:47:19.910 --> 00:47:23.370 these days than it was 20 years ago. 00:47:23.370 --> 00:47:25.400 I just want to point out where this system-- 00:47:25.400 --> 00:47:28.790 how it was discovered and where it came from. 00:47:28.790 --> 00:47:30.440 And I like this as an example. 00:47:30.440 --> 00:47:34.280 Much like for the fly genes that defined 00:47:34.280 --> 00:47:37.370 major signaling pathways, this is a discovery 00:47:37.370 --> 00:47:40.760 that came from fundamental research 00:47:40.760 --> 00:47:46.100 on, basically, the ecology of bacteria. 00:47:46.100 --> 00:47:50.060 So this CRISPR-Cas9 system essentially evolved 00:47:50.060 --> 00:47:54.920 in bacteria as a form of an arms race between bacteria 00:47:54.920 --> 00:47:57.260 and their predators, bacteriophage, 00:47:57.260 --> 00:48:00.900 which are viruses that infect bacteria. 00:48:00.900 --> 00:48:09.410 So this is an arms race between bacteria 00:48:09.410 --> 00:48:12.020 and their vicious predators, bacteriophage. 00:48:18.560 --> 00:48:22.400 And what CRISPR is, where these enzymes and this system 00:48:22.400 --> 00:48:27.260 evolved from, is this is a form of an adaptive immune system 00:48:27.260 --> 00:48:31.310 for bacteria, which is pretty wild in and of itself. 00:48:31.310 --> 00:48:36.965 So CRISPR is an adaptive immune system for bacteria. 00:48:41.870 --> 00:48:45.170 If you haven't gotten your flu shot, you should get it. 00:48:45.170 --> 00:48:50.580 We'll talk about human immunity later in the semester. 00:48:50.580 --> 00:48:54.550 But this is where bacterial immunity kind of-- 00:48:54.550 --> 00:48:56.570 I'm sneaking it in. 00:48:56.570 --> 00:48:59.910 So the way this works in bacteria-- 00:48:59.910 --> 00:49:03.110 what CRISPR stands for is Clusters 00:49:03.110 --> 00:49:07.710 of Regularly Interspaced Short Palindromic Repeats. 00:49:07.710 --> 00:49:11.060 So you can see already thank god they gave it an acronym. 00:49:11.060 --> 00:49:14.160 Otherwise, it wouldn't be getting nearly as much buzz, 00:49:14.160 --> 00:49:16.340 because no one can say that. 00:49:16.340 --> 00:49:20.270 And so where this CRISPR came from is 00:49:20.270 --> 00:49:24.660 on bacterial chromosomes of many bacteria, 00:49:24.660 --> 00:49:28.610 there's these clusters of interspaced short palindromic 00:49:28.610 --> 00:49:33.240 repeats, and the repeats are interrupted by spacers. 00:49:33.240 --> 00:49:36.780 And what researchers discovered are these spacers 00:49:36.780 --> 00:49:41.190 have sequence similarity and identity to sequences 00:49:41.190 --> 00:49:44.970 that are from bacteriophage. 00:49:44.970 --> 00:49:46.920 So each of these colored sequences 00:49:46.920 --> 00:49:51.000 here has some type of complementarity 00:49:51.000 --> 00:49:54.510 to some type of bacteriophage. 00:49:54.510 --> 00:49:59.310 And so when a phage infects bacteria, or some bacteria, 00:49:59.310 --> 00:50:01.890 what happens is that there's a system 00:50:01.890 --> 00:50:05.370 to recognize that foreign genetic element 00:50:05.370 --> 00:50:09.160 and take a piece of it and insert it in the genome. 00:50:09.160 --> 00:50:11.190 And that serves as a memory for the bacteria 00:50:11.190 --> 00:50:16.200 to remember that it got infected by that particular phage. 00:50:16.200 --> 00:50:18.000 And then, later on, what the bacteria 00:50:18.000 --> 00:50:21.570 does is it transcribes this region 00:50:21.570 --> 00:50:25.950 and forms these mature what are known as CRISPR RNAs, where you 00:50:25.950 --> 00:50:28.920 can see there's some sequence would recognize 00:50:28.920 --> 00:50:31.210 a foreign genetic element. 00:50:31.210 --> 00:50:36.030 So, therefore, in the future, if this phage came around again, 00:50:36.030 --> 00:50:39.450 what would happen is one of these CRISPR RNAs 00:50:39.450 --> 00:50:42.450 would recognize the foreign genetic element 00:50:42.450 --> 00:50:45.030 through base pair complementarity, 00:50:45.030 --> 00:50:46.830 and it would know to cut it. 00:50:46.830 --> 00:50:49.050 And after the target is cut, it's 00:50:49.050 --> 00:50:51.930 then degraded by the bacterial cell. 00:50:51.930 --> 00:50:56.220 So this is a way for bacteria to remember what viruses 00:50:56.220 --> 00:51:01.260 have infected them and to have a defense mechanism against it. 00:51:01.260 --> 00:51:03.810 So it's a pretty cool system. 00:51:03.810 --> 00:51:05.670 You know, what's also cool about this system 00:51:05.670 --> 00:51:09.720 is it's an adaptive immune system, similar to how we sort 00:51:09.720 --> 00:51:13.140 of recognize foreign pathogens. 00:51:13.140 --> 00:51:15.810 What's different about it is this is heritable. 00:51:15.810 --> 00:51:18.120 It's incorporated into the genome. 00:51:18.120 --> 00:51:21.300 And the more phage the descendants of this bacteria 00:51:21.300 --> 00:51:24.510 see, the more of these repeats you see. 00:51:24.510 --> 00:51:28.080 So this is a heritable immune system, which, unfortunately, 00:51:28.080 --> 00:51:30.630 we don't have. 00:51:30.630 --> 00:51:32.280 So you should still get your flu shot. 00:51:35.970 --> 00:51:38.010 We'll talk about vaccination later on. 00:51:40.680 --> 00:51:44.480 Have a good few days, and I will see you on Friday.