WEBVTT 00:00:16.710 --> 00:00:19.980 PROFESSOR: And today I'm going to talk about DNA sequencing. 00:00:19.980 --> 00:00:24.510 And I want to start by just sort of illustrating 00:00:24.510 --> 00:00:29.830 an example of how knowing the DNA sequence can be helpful. 00:00:29.830 --> 00:00:33.310 So you remember in the last lecture, 00:00:33.310 --> 00:00:37.050 we talked about how one might identify 00:00:37.050 --> 00:00:41.730 a gene through functional complementation. 00:00:41.730 --> 00:00:46.950 And this process involved making a DNA library that 00:00:46.950 --> 00:00:52.750 had different fragments of DNA cloned into different plasmids 00:00:52.750 --> 00:00:56.770 and then involved finding the needle in the haystack where 00:00:56.770 --> 00:01:01.200 you find the gene that can rescue a defect in a mutant 00:01:01.200 --> 00:01:01.920 that you have. 00:01:05.620 --> 00:01:15.500 So if this line that I'm drawing here is genomic DNA, 00:01:15.500 --> 00:01:19.820 and it could be genomic DNA from, 00:01:19.820 --> 00:01:25.700 let's say, a prototroph for LEU2, the leucine gene. 00:01:25.700 --> 00:01:27.380 So this is from a prototroph. 00:01:31.650 --> 00:01:38.140 Then you could cut up the DNA with EcoRI. 00:01:38.140 --> 00:01:41.460 And if there is not a restriction site in this LEU2 00:01:41.460 --> 00:01:46.710 gene, you get a fragment that contains the LEU2 gene. 00:01:46.710 --> 00:01:53.100 And then you could clone this into some type of plasmid 00:01:53.100 --> 00:01:56.750 that replicates in the organism that you're introducing 00:01:56.750 --> 00:01:59.910 it and propagating it in. 00:01:59.910 --> 00:02:04.590 And so that would allow you to then test whether or not 00:02:04.590 --> 00:02:10.750 this piece of DNA that you have compliments a LEU2 auxotroph, 00:02:10.750 --> 00:02:11.250 OK? 00:02:12.240 --> 00:02:14.310 Now one thing I want to point out 00:02:14.310 --> 00:02:23.190 is that because these EcoR1 sites, these sticky ends, 00:02:23.190 --> 00:02:28.230 would recognize this EcoR1 one end or this EcoR1 end, 00:02:28.230 --> 00:02:30.780 you can imagine that this gene-- 00:02:30.780 --> 00:02:33.330 if the gene reads this way to this way-- 00:02:33.330 --> 00:02:36.420 it could insert this way into the plasmid. 00:02:36.420 --> 00:02:39.340 Or it could insert in the opposite direction. 00:02:39.340 --> 00:02:42.120 So it could be inverted. 00:02:42.120 --> 00:02:45.270 So this would have some sort of origin of replication 00:02:45.270 --> 00:02:48.960 and some type of selectable marker. 00:02:48.960 --> 00:02:51.540 But if you have the same restriction site 00:02:51.540 --> 00:02:54.210 it can insert one way or the opposite way. 00:02:54.210 --> 00:02:58.680 That's just one thing I wanted to point out. 00:02:58.680 --> 00:03:02.290 Now let's say rather than leucine, 00:03:02.290 --> 00:03:05.160 you're interested in cycling dependent kinase, 00:03:05.160 --> 00:03:09.000 and you had a mutant end CDK and you had this sequence 00:03:09.000 --> 00:03:11.880 of your yeast CDK gene. 00:03:11.880 --> 00:03:18.240 Well, rather than having to dig through a whole library 00:03:18.240 --> 00:03:22.530 of pieces of DNA for the CDK gene, 00:03:22.530 --> 00:03:24.930 basically you're sort of fishing for that needle 00:03:24.930 --> 00:03:26.190 in the haystack. 00:03:26.190 --> 00:03:29.010 If you knew the sequence of the human genome, 00:03:29.010 --> 00:03:33.850 you'd be able to identify similar genes by sequence 00:03:33.850 --> 00:03:34.980 homology. 00:03:34.980 --> 00:03:37.860 And you could then take a more direct approach, 00:03:37.860 --> 00:03:39.600 where you take-- 00:03:39.600 --> 00:03:42.360 let's say you have a piece of human DNA 00:03:42.360 --> 00:03:48.930 now, double stranded DNA, and it has the CDK gene. 00:03:48.930 --> 00:03:53.160 You could take human DNA with this CDK gene. 00:03:53.160 --> 00:03:56.520 And you have unique sequence around the CDK 00:03:56.520 --> 00:04:00.885 gene, which would allow you to denature this DNA. 00:04:04.180 --> 00:04:11.010 And if you denature the DNA, you'd 00:04:11.010 --> 00:04:12.915 get two single strands of DNA. 00:04:16.260 --> 00:04:18.240 And you could then design primers 00:04:18.240 --> 00:04:23.350 that recognize unique sequences flanking the CDK gene. 00:04:23.350 --> 00:04:26.700 So you could imagine you'd have a primer here 00:04:26.700 --> 00:04:28.590 and a primer here. 00:04:28.590 --> 00:04:32.670 And then you could use PCR to amplify specifically 00:04:32.670 --> 00:04:38.730 CDK gene from, it could be the genome or from some library. 00:04:38.730 --> 00:04:45.960 And then you get this fragment here, which includes CDK. 00:04:45.960 --> 00:04:49.230 So knowing the sequence of the genome 00:04:49.230 --> 00:04:54.390 would allow you to more rapidly go from maybe a gene 00:04:54.390 --> 00:04:57.720 that you've identified as being important in one organism, 00:04:57.720 --> 00:05:01.650 and find the human equivalent that might be doing something 00:05:01.650 --> 00:05:05.280 similar in humans. 00:05:05.280 --> 00:05:07.230 So this step here is basically PCR. 00:05:10.530 --> 00:05:14.820 And let's say the CDK gene had restriction sites. 00:05:14.820 --> 00:05:22.350 Let's see, we'll say restriction site K and A here. 00:05:22.350 --> 00:05:24.420 Then if you have these restriction sites 00:05:24.420 --> 00:05:27.360 in your fragment of DNA, you can then 00:05:27.360 --> 00:05:31.200 digest or cut that piece of DNA with these restriction 00:05:31.200 --> 00:05:33.570 endonucleases. 00:05:33.570 --> 00:05:37.740 And then you'd get a fragment of CDK 00:05:37.740 --> 00:05:41.820 that has K and A sticky ends. 00:05:41.820 --> 00:05:45.420 We'll pretend that both of these have sticky ends. 00:05:45.420 --> 00:05:49.935 And now you have unique sticky ends between K and A. 00:05:49.935 --> 00:05:58.740 And you might have a vector that also has these two sites. 00:05:58.740 --> 00:06:02.510 And you could digest this vector with these two enzymes. 00:06:02.510 --> 00:06:06.890 And that would allow you to insert the specific gene 00:06:06.890 --> 00:06:09.920 in this plasmid. 00:06:09.920 --> 00:06:12.350 And if you have two unique sites, 00:06:12.350 --> 00:06:18.830 because K only recognizes K here and A only recognizes A, 00:06:18.830 --> 00:06:20.690 then it will ligate in. 00:06:20.690 --> 00:06:23.540 But you can do it with a specific orientation 00:06:23.540 --> 00:06:26.810 because you have two different restriction sites. 00:06:26.810 --> 00:06:32.360 So I hope you all see how it's with one restriction 00:06:32.360 --> 00:06:33.590 site versus two. 00:06:39.180 --> 00:06:39.680 All right. 00:06:39.680 --> 00:06:42.230 Now let's say you want to do something more complicated 00:06:42.230 --> 00:06:44.270 than this. 00:06:44.270 --> 00:06:49.310 Let's say rather than just identifying the gene that's 00:06:49.310 --> 00:06:54.500 involved in cell division, you want to engineer a new gene, 00:06:54.500 --> 00:06:59.210 in order to determine where this particular protein, CDK, 00:06:59.210 --> 00:07:03.140 localizes in the cell. 00:07:03.140 --> 00:07:09.470 So we have CDK, which could be from yeast or human, 00:07:09.470 --> 00:07:11.290 it doesn't matter. 00:07:11.290 --> 00:07:17.090 And you want to engineer a new protein, basically, 00:07:17.090 --> 00:07:19.790 that you can see. 00:07:19.790 --> 00:07:22.670 So remember Professor Imperiali introduced 00:07:22.670 --> 00:07:26.360 green fluorescent protein earlier in the year. 00:07:26.360 --> 00:07:28.700 And this green fluorescent protein 00:07:28.700 --> 00:07:34.050 is from a gene from jellyfish. 00:07:34.050 --> 00:07:37.700 So now we could, using what I've told you, 00:07:37.700 --> 00:07:40.580 reconstruct or engineer a gene that 00:07:40.580 --> 00:07:43.130 has DNA from three different organisms, 00:07:43.130 --> 00:07:46.910 in order to make a CDK variant that we 00:07:46.910 --> 00:07:49.650 are able to see in the cell. 00:07:49.650 --> 00:07:53.750 So remember, a green fluorescent protein is like a beacon, 00:07:53.750 --> 00:07:55.700 if it's attached to a protein. 00:07:55.700 --> 00:07:58.460 If you shine blue light on it, it emits green light. 00:07:58.460 --> 00:08:04.220 And so you can use a fluorescent microscope in order to see it. 00:08:04.220 --> 00:08:07.240 In this case, let's say there's also another restriction site 00:08:07.240 --> 00:08:13.730 here, R. And let's say you have a fragment of GFP that 00:08:13.730 --> 00:08:17.630 has two restriction sites, A and R. You could then 00:08:17.630 --> 00:08:20.990 cut this fragment and this fragment 00:08:20.990 --> 00:08:24.050 with these restriction enzymes A and R. 00:08:24.050 --> 00:08:30.500 And you could insert GFP at the C terminus of the CDK gene. 00:08:30.500 --> 00:08:38.720 So you could go and have a gene that has CDK GFP inserted 00:08:38.720 --> 00:08:42.169 inside a bacterial vector. 00:08:42.169 --> 00:08:45.680 Now which one of these junction sites 00:08:45.680 --> 00:08:47.840 do you think would be most sensitive in doing 00:08:47.840 --> 00:08:49.130 this type of experiment? 00:08:51.780 --> 00:08:54.060 So there are three junction sites. 00:08:54.060 --> 00:08:56.447 There's this one, this one, and this one. 00:08:56.447 --> 00:08:58.030 Which is the one you're probably going 00:08:58.030 --> 00:08:59.870 to put the most thought into when 00:08:59.870 --> 00:09:02.000 you're doing this experiment? 00:09:02.000 --> 00:09:04.676 Yes, Miles. 00:09:04.676 --> 00:09:06.450 AUDIENCE: The A? 00:09:06.450 --> 00:09:08.000 ADAM MARTIN: The A site. 00:09:08.000 --> 00:09:10.160 Miles is exactly right. 00:09:10.160 --> 00:09:11.750 This one is going to be important. 00:09:11.750 --> 00:09:15.744 And why did you choose that site? 00:09:15.744 --> 00:09:18.100 AUDIENCE: Of the three sites, two 00:09:18.100 --> 00:09:24.874 are half insert, half originals [INAUDIBLE].. 00:09:24.874 --> 00:09:29.360 But at A, both sides of it are inserts. 00:09:29.360 --> 00:09:31.825 So [INAUDIBLE] carefully. 00:09:31.825 --> 00:09:33.200 ADAM MARTIN: And if you're trying 00:09:33.200 --> 00:09:35.540 to make a fusion protein, what's going 00:09:35.540 --> 00:09:39.900 to be an important quality of this? 00:09:39.900 --> 00:09:41.920 Malik, DID you have a point? 00:09:41.920 --> 00:09:49.435 AUDIENCE: Well, they try to [INAUDIBLE] 00:09:49.435 --> 00:09:52.369 we'd have to make sure that the [INAUDIBLE].. 00:09:56.935 --> 00:09:58.060 ADAM MARTIN: Excellent job. 00:09:58.060 --> 00:10:01.450 So Malik just pointed out two really important things. 00:10:01.450 --> 00:10:04.330 To make this a fusion protein, you 00:10:04.330 --> 00:10:06.940 have two different open reading frames. 00:10:06.940 --> 00:10:08.920 These two open reading frames have 00:10:08.920 --> 00:10:11.410 to be in frame with each other. 00:10:11.410 --> 00:10:15.850 So this junction here has to be in frame where 00:10:15.850 --> 00:10:19.060 GFP is in frame with CDK, meaning 00:10:19.060 --> 00:10:26.080 that you're reading the same triplet codons for GFP, 00:10:26.080 --> 00:10:28.450 there in the same frame as CDK. 00:10:28.450 --> 00:10:31.090 Also, you want to make sure there's no stop codon here. 00:10:34.200 --> 00:10:36.310 Because if you had a stop codon here, 00:10:36.310 --> 00:10:38.650 you're just going to make a CDK protein. 00:10:38.650 --> 00:10:40.510 And then it's going to stop and then 00:10:40.510 --> 00:10:42.260 you won't have it fused to GFP. 00:10:46.440 --> 00:10:50.080 And you guys will work through more of these in the homework. 00:10:50.080 --> 00:10:53.200 So you'll be able to get a sense of it. 00:10:53.200 --> 00:10:55.870 So now for the remainder of this lecture 00:10:55.870 --> 00:10:58.750 and also for Monday's lecture, I want 00:10:58.750 --> 00:11:01.840 to go through a problem with you. 00:11:01.840 --> 00:11:06.580 Basically, if you have a given disease that's heritable, 00:11:06.580 --> 00:11:11.380 how might you go from knowing that disease 00:11:11.380 --> 00:11:15.640 is heritable to finding out what gene 00:11:15.640 --> 00:11:18.940 is responsible for that given disease? 00:11:18.940 --> 00:11:20.740 And this is going to involve thinking 00:11:20.740 --> 00:11:25.820 about different levels of resolution, in terms of maps. 00:11:25.820 --> 00:11:28.480 So the highest resolution map you can have 00:11:28.480 --> 00:11:31.780 for a genome is the sequence. 00:11:31.780 --> 00:11:37.120 You can have the full nucleotide sequence of a genome. 00:11:37.120 --> 00:11:39.220 And that's the highest possible resolution 00:11:39.220 --> 00:11:40.960 because you have single nucleotide 00:11:40.960 --> 00:11:46.540 resolution as to what every single base pair is. 00:11:46.540 --> 00:11:50.320 But that's like knowing like your apartment 00:11:50.320 --> 00:11:54.010 number and your street number and basically knowing 00:11:54.010 --> 00:11:55.300 everything. 00:11:55.300 --> 00:11:59.080 But starting out, you might want to know what continent it's on, 00:11:59.080 --> 00:12:02.050 or what country is it in. 00:12:02.050 --> 00:12:07.300 And so you first have to narrow down the possible locations 00:12:07.300 --> 00:12:09.520 for a given disease gene. 00:12:09.520 --> 00:12:14.080 And that will, at first, involve establishing what chromosome 00:12:14.080 --> 00:12:17.970 and what region of a chromosome a given disease 00:12:17.970 --> 00:12:20.710 allele is linked to. 00:12:20.710 --> 00:12:29.860 And that involves making essentially a linkage map, 00:12:29.860 --> 00:12:35.290 where you establish where a disease gene is located based 00:12:35.290 --> 00:12:40.960 on its linkage to known markers that are present in the genome. 00:12:40.960 --> 00:12:46.930 Now this is going to require that you remember back 00:12:46.930 --> 00:12:49.600 two weeks ago, to when we talked about linkage 00:12:49.600 --> 00:12:51.280 and recombination. 00:12:51.280 --> 00:12:57.970 And you'll recall that we were looking 00:12:57.970 --> 00:13:05.110 at the linkage between genes and flies and genes and yeast. 00:13:05.110 --> 00:13:09.640 One difference between that type of linkage mapping 00:13:09.640 --> 00:13:12.100 and human linkage mapping is we don't 00:13:12.100 --> 00:13:17.950 have really clear traits that are defined by single genes. 00:13:17.950 --> 00:13:22.660 You can't just take hair color and map the hair color gene 00:13:22.660 --> 00:13:26.180 to link it to a disease gene. 00:13:26.180 --> 00:13:30.740 Because hair color is determined by many, many different genes. 00:13:30.740 --> 00:13:32.740 So in fruit flies, you can take white eyes 00:13:32.740 --> 00:13:35.620 and see if it's connected with yellow body color 00:13:35.620 --> 00:13:39.790 because both of those are determined by single genes. 00:13:39.790 --> 00:13:44.590 So we need something other than just having phenotypic traits 00:13:44.590 --> 00:13:46.150 that we can track. 00:13:46.150 --> 00:13:49.330 We need what are known as molecular markers 00:13:49.330 --> 00:13:51.760 to be able to perform linkage mapping. 00:13:56.770 --> 00:14:00.070 And so what we need in these molecular markers-- 00:14:00.070 --> 00:14:02.530 well, if we just think about if we 00:14:02.530 --> 00:14:09.490 wanted to determine the linkage between the A and B genes. 00:14:09.490 --> 00:14:11.290 And if you did this cross, would you 00:14:11.290 --> 00:14:12.655 be able to determine linkage? 00:14:19.480 --> 00:14:23.050 Georgia, you made a motion that was correct. 00:14:23.050 --> 00:14:25.030 Tell me. 00:14:25.030 --> 00:14:27.030 Why did you shake your head no? 00:14:27.030 --> 00:14:30.110 AUDIENCE: They'd all be heterozygous. 00:14:30.110 --> 00:14:33.100 ADAM MARTIN: Yeah they'd all be heterozygous. 00:14:33.100 --> 00:14:37.420 Because this individual has the same allele 00:14:37.420 --> 00:14:39.460 on both chromosomes, you're not going 00:14:39.460 --> 00:14:43.690 to be able to differentiate one chromosome from the other. 00:14:43.690 --> 00:14:45.610 And so the point I want to make is 00:14:45.610 --> 00:14:49.450 that in order to see linkage, what you need is variation. 00:14:54.360 --> 00:14:56.850 So we need to have variation. 00:14:56.850 --> 00:15:01.650 And another term for genetic variation is polymorphism. 00:15:01.650 --> 00:15:11.310 So we need polymorphism, or genetic variation, 00:15:11.310 --> 00:15:13.320 between these molecular markers. 00:15:16.530 --> 00:15:21.150 We also need genetic variation in the disease. 00:15:21.150 --> 00:15:22.380 But we have that. 00:15:22.380 --> 00:15:25.560 We have individuals that are affected 00:15:25.560 --> 00:15:27.180 by a disease and individuals that 00:15:27.180 --> 00:15:28.590 are not affected by a disease. 00:15:28.590 --> 00:15:31.740 So we have variation in alleles there. 00:15:31.740 --> 00:15:36.480 But in order to map it with a molecular marker, 00:15:36.480 --> 00:15:38.850 to map linkage to a molecular marker, 00:15:38.850 --> 00:15:41.520 you also need variation here. 00:15:41.520 --> 00:15:43.890 So the problem with this cross is here 00:15:43.890 --> 00:15:47.370 you need to have heterozygote. 00:15:47.370 --> 00:15:53.160 There needs to be variation in this individual, where 00:15:53.160 --> 00:15:57.930 both of these alleles are heterozygous. 00:15:57.930 --> 00:16:01.680 So now I want to talk about some of these molecular markers 00:16:01.680 --> 00:16:08.700 that we can use, and how they vary between individuals 00:16:08.700 --> 00:16:10.050 and between chromosomes. 00:16:12.630 --> 00:16:15.450 Now this is going to be maybe the lowest resolution map. 00:16:15.450 --> 00:16:18.990 But I'm talking about this linkage map here. 00:16:18.990 --> 00:16:21.780 And you can see highlighted that the bottom here 00:16:21.780 --> 00:16:24.450 are various types of polymorphisms 00:16:24.450 --> 00:16:28.380 that we can use to link a given disease 00:16:28.380 --> 00:16:32.340 allele to a specific chromosome and a specific place 00:16:32.340 --> 00:16:33.280 on chromosome. 00:16:36.120 --> 00:16:45.310 So I'll start with the first one, which is a simple sequence 00:16:45.310 --> 00:16:46.510 repeat. 00:16:46.510 --> 00:16:47.860 It goes by many names. 00:16:47.860 --> 00:16:50.035 But I will stick with what's on the slide. 00:16:54.020 --> 00:17:00.280 So a simple sequence repeat is also known as a microsatellite. 00:17:00.280 --> 00:17:04.690 So you might see that term floating around, 00:17:04.690 --> 00:17:06.880 if you're reading about this. 00:17:06.880 --> 00:17:12.290 And what a simple sequence repeat is, as the name implies, 00:17:12.290 --> 00:17:14.230 it's a simple sequence. 00:17:14.230 --> 00:17:17.560 It could be a dinucleotide, like CA. 00:17:17.560 --> 00:17:19.390 And it's just a dinucleotide that's 00:17:19.390 --> 00:17:23.050 repeated over and over again. 00:17:23.050 --> 00:17:26.560 So on a chromosome, you might have a unique sequence, 00:17:26.560 --> 00:17:28.900 which I'll just draw as a line. , 00:17:28.900 --> 00:17:33.160 And then you could have a CA dinucleotide that's repeated 00:17:33.160 --> 00:17:37.570 some number of times, N. And then that's followed by another 00:17:37.570 --> 00:17:40.480 unique sequence. 00:17:40.480 --> 00:17:41.900 And that's what's present in it. 00:17:41.900 --> 00:17:43.870 So that would be one strand. 00:17:43.870 --> 00:17:45.850 And then in the opposite strand, you'd 00:17:45.850 --> 00:17:49.360 have a unique sequence, the complement of CA, 00:17:49.360 --> 00:17:52.140 which is GT, and then, again, unique sequence. 00:17:54.790 --> 00:18:00.040 And so there's variation in the number of repeats of the CA. 00:18:00.040 --> 00:18:02.530 And so there's polymorphism. 00:18:02.530 --> 00:18:05.980 So we can use this to establish linkage 00:18:05.980 --> 00:18:08.890 between this marker and a phenotype, 00:18:08.890 --> 00:18:12.250 like a disease phenotype. 00:18:12.250 --> 00:18:15.820 So how might you detect the number of repeats 00:18:15.820 --> 00:18:17.440 that are present here? 00:18:17.440 --> 00:18:20.140 Anyone have an idea of a tool that we've discussed 00:18:20.140 --> 00:18:21.280 that could be used here? 00:18:24.060 --> 00:18:28.340 So one hint that I gave you is that the sequence here 00:18:28.340 --> 00:18:30.930 is unique and the sequence here is unique. 00:18:30.930 --> 00:18:34.160 So is there a way we can leverage that unique sequence 00:18:34.160 --> 00:18:36.058 to determine whether there's a difference 00:18:36.058 --> 00:18:37.100 in the number of repeats? 00:18:42.500 --> 00:18:46.860 What's a technique we discussed that involves 00:18:46.860 --> 00:18:51.530 some component of the technique recognizing a unique sequence? 00:18:51.530 --> 00:18:52.140 Yeah, Natalie? 00:18:52.140 --> 00:18:53.790 AUDIENCE: CRISPR Cas9. 00:18:53.790 --> 00:18:57.720 ADAM MARTIN: Well, CRISPR Cas9 is a possibility. 00:18:57.720 --> 00:18:59.860 Jeremy, did you have an idea? 00:18:59.860 --> 00:19:00.740 AUDIENCE: PCR? 00:19:00.740 --> 00:19:03.510 ADAM MARTIN: PCR-- so it's true. 00:19:03.510 --> 00:19:05.770 You could get it to recognize that. 00:19:05.770 --> 00:19:08.520 But then you have to detect it, somehow. 00:19:08.520 --> 00:19:12.510 So what's more commonly used is PCR. 00:19:12.510 --> 00:19:15.090 Those are both good ideas. 00:19:15.090 --> 00:19:18.780 But using PCR, you could design a primer here 00:19:18.780 --> 00:19:20.460 and a primer here. 00:19:20.460 --> 00:19:24.000 And you could amplify this repeat sequence. 00:19:24.000 --> 00:19:26.430 And the number of repeats would determine 00:19:26.430 --> 00:19:30.000 the size of your PCR fragment. 00:19:30.000 --> 00:19:35.970 So if you did PCR, then you'd get a PCR fragment that 00:19:35.970 --> 00:19:41.130 has the primers on each end, but then has this certain size 00:19:41.130 --> 00:19:44.130 based on the number of repeats. 00:19:44.130 --> 00:19:46.680 So in that case, we need some sort of tool 00:19:46.680 --> 00:19:52.380 that enables us to determine the size of a particular DNA 00:19:52.380 --> 00:19:54.090 fragment. 00:19:54.090 --> 00:19:57.270 And so I'm going to just introduce to you one such tool, 00:19:57.270 --> 00:20:00.870 which is gel electrophoresis. 00:20:00.870 --> 00:20:04.110 And gel electrophoresis involves taking DNA 00:20:04.110 --> 00:20:08.400 that you've generated, by either PCR or by cutting up 00:20:08.400 --> 00:20:13.320 DNA with a restriction enzyme, and loading it 00:20:13.320 --> 00:20:16.680 in a gel that has agarose. 00:20:16.680 --> 00:20:18.150 Maybe it's composed of agarose. 00:20:18.150 --> 00:20:20.970 It could be composed of polyacrylamide. 00:20:20.970 --> 00:20:24.360 And then because DNA is negatively charged, 00:20:24.360 --> 00:20:28.380 the backbone, if you run a current through it, 00:20:28.380 --> 00:20:31.290 such as the positive electrode is at the bottom, 00:20:31.290 --> 00:20:36.540 then the DNA is going to snake through this gel. 00:20:36.540 --> 00:20:38.460 Now we'll do a quick demonstration, 00:20:38.460 --> 00:20:39.510 if you two could come up. 00:20:39.510 --> 00:20:41.520 I need one volunteer. 00:20:41.520 --> 00:20:44.580 Ori, find 10 of your friends and bring them down. 00:20:56.230 --> 00:20:56.730 All right. 00:20:56.730 --> 00:20:58.670 That's probably good. 00:20:58.670 --> 00:21:01.250 Yeah. 00:21:01.250 --> 00:21:03.680 All right, Hannah, why don't you-- 00:21:03.680 --> 00:21:05.260 you guys have to link up, OK? 00:21:08.300 --> 00:21:08.950 Stay over here. 00:21:08.950 --> 00:21:09.950 We'll start at this end. 00:21:09.950 --> 00:21:12.800 This is the negative electrode over here. 00:21:12.800 --> 00:21:15.950 The positive electrode is going to be down there. 00:21:15.950 --> 00:21:19.550 And Jackie is going to be our single nucleotide. 00:21:19.550 --> 00:21:22.220 You guys link like-- 00:21:22.220 --> 00:21:23.580 yeah. 00:21:23.580 --> 00:21:27.080 You don't have to do-si-do, or anything like that. 00:21:27.080 --> 00:21:28.010 All right. 00:21:28.010 --> 00:21:29.750 Now what I want you guys to do is 00:21:29.750 --> 00:21:32.570 I want you to slalom through these cones 00:21:32.570 --> 00:21:34.250 like it's all agarose gel. 00:21:34.250 --> 00:21:37.430 So that you're going towards the other side. 00:21:37.430 --> 00:21:39.680 And I'm going to turn on the current now. 00:21:39.680 --> 00:21:40.220 So go. 00:21:47.330 --> 00:21:49.590 All right, stop. 00:21:49.590 --> 00:21:50.090 All right. 00:21:50.090 --> 00:21:53.090 See how the shorter DNA fragment is 00:21:53.090 --> 00:21:55.790 able to more easily navigate through the cones 00:21:55.790 --> 00:21:58.370 and get farther. 00:21:58.370 --> 00:22:01.770 So it was somewhat rigged. 00:22:01.770 --> 00:22:02.270 I know. 00:22:02.270 --> 00:22:05.900 But I just needed some way to make sure you always 00:22:05.900 --> 00:22:09.050 remember that the shorter nucleotide, or the shorter 00:22:09.050 --> 00:22:11.480 fragment, is going to migrate faster. 00:22:11.480 --> 00:22:12.830 You guys can go back up. 00:22:12.830 --> 00:22:15.120 Thank you for your participation. 00:22:15.120 --> 00:22:16.610 Let's give them round of applause. 00:22:16.610 --> 00:22:20.068 [APPLAUSE] 00:22:22.040 --> 00:22:22.540 All right. 00:22:22.540 --> 00:22:27.660 So what you just saw is that the longer DNA fragments, 00:22:27.660 --> 00:22:31.540 they're going to be more inhibited by moving 00:22:31.540 --> 00:22:32.710 through the gel. 00:22:32.710 --> 00:22:34.960 And so they're going to move slower and thus, 00:22:34.960 --> 00:22:36.827 not move as far in the gel. 00:22:36.827 --> 00:22:38.410 Whereas, the small fragments are going 00:22:38.410 --> 00:22:40.600 to move much faster because they're 00:22:40.600 --> 00:22:43.870 able to maneuver their way through this gel 00:22:43.870 --> 00:22:45.710 much more quickly. 00:22:45.710 --> 00:22:49.060 So there's going to be an inverse proportionality 00:22:49.060 --> 00:22:55.840 between the size of the DNA chain and its rate of movement. 00:22:55.840 --> 00:22:58.240 You're always going to see the shorter DNA 00:22:58.240 --> 00:23:00.190 fragment moving faster. 00:23:00.190 --> 00:23:01.990 So what one of these gels actually 00:23:01.990 --> 00:23:05.120 looks like is shown here. 00:23:05.120 --> 00:23:08.260 So this is a DNA gel that's agarose. 00:23:08.260 --> 00:23:11.890 And DNA has been run in these different samples. 00:23:11.890 --> 00:23:14.140 And what you're seeing is this gel 00:23:14.140 --> 00:23:16.480 is subsequently stained with a dye, 00:23:16.480 --> 00:23:20.080 like ethidium bromide, which allows you to visualize 00:23:20.080 --> 00:23:22.690 the individual DNA fragments. 00:23:22.690 --> 00:23:26.080 And so a band on this gel indicates a whole bunch 00:23:26.080 --> 00:23:30.100 of DNA fragments that are all roughly the same length. 00:23:30.100 --> 00:23:32.740 So essentially, you can measure DNA length 00:23:32.740 --> 00:23:34.270 using this technique. 00:23:34.270 --> 00:23:36.530 What's over here at the end of the gel, 00:23:36.530 --> 00:23:39.010 this is probably some sort of DNA ladder, 00:23:39.010 --> 00:23:41.470 where you have DNA fragments of known length 00:23:41.470 --> 00:23:44.230 that you can use to calibrate the length 00:23:44.230 --> 00:23:47.980 of these bands over here. 00:23:47.980 --> 00:23:50.520 So this is how you measure DNA length. 00:23:50.520 --> 00:23:52.540 And we're going to use it over and over again, 00:23:52.540 --> 00:23:56.440 as we talk about DNA and sequencing. 00:23:56.440 --> 00:23:58.360 So now, let's think about how this 00:23:58.360 --> 00:24:01.480 is going to help us establish linkage 00:24:01.480 --> 00:24:03.880 between a particular marker in the genome 00:24:03.880 --> 00:24:06.920 and a genetic disease. 00:24:06.920 --> 00:24:10.330 So if we think about these microsatellite repeats, 00:24:10.330 --> 00:24:11.890 I told you they're polymorphic. 00:24:11.890 --> 00:24:15.400 They exhibit a lot of variation in size. 00:24:15.400 --> 00:24:17.590 And so here's an example showing you 00:24:17.590 --> 00:24:21.940 a female who has two intermediate sized 00:24:21.940 --> 00:24:23.500 microsatellites. 00:24:23.500 --> 00:24:25.540 And if you look at this-- 00:24:25.540 --> 00:24:28.660 if you did PCR and measured the size of these, 00:24:28.660 --> 00:24:30.660 you get two different bands because there 00:24:30.660 --> 00:24:34.690 are two different alleles of different length here. 00:24:34.690 --> 00:24:40.120 So you can see this individual has two intermediate length 00:24:40.120 --> 00:24:41.420 repeats. 00:24:41.420 --> 00:24:45.100 And this person has had children with an individual 00:24:45.100 --> 00:24:48.490 that has a short and a long microsatellite. 00:24:48.490 --> 00:24:51.640 And you can see that on the gel, here. 00:24:51.640 --> 00:24:55.750 Now this female is affected by some disease. 00:24:55.750 --> 00:24:59.690 And these two individuals have children. 00:24:59.690 --> 00:25:02.500 And you can see that a number of those children 00:25:02.500 --> 00:25:04.460 are affected by the disease. 00:25:04.460 --> 00:25:06.820 So what mode of inheritance does this look like? 00:25:10.850 --> 00:25:14.000 If you had your choice between autosomal recessive, 00:25:14.000 --> 00:25:16.690 autosomal dominant, sex linked dominant, 00:25:16.690 --> 00:25:22.460 and sex linked recessive, what mode of inheritance 00:25:22.460 --> 00:25:23.480 is this looking like? 00:25:26.570 --> 00:25:27.080 Oh, Carmen. 00:25:27.080 --> 00:25:29.200 AUDIENCE: Autosomal recessive. 00:25:29.200 --> 00:25:31.550 ADAM MARTIN: Autosomal recessive? 00:25:31.550 --> 00:25:35.900 Why do you go with recessive? 00:25:35.900 --> 00:25:37.010 Yeah, go ahead. 00:25:37.010 --> 00:25:40.490 AUDIENCE: Because there is a male that's affected. 00:25:44.770 --> 00:25:47.895 But not both of the parents are affected. 00:25:47.895 --> 00:25:55.220 So it seems like the father is heterozygous 00:25:55.220 --> 00:25:57.232 and the mother is homozygous recessive. 00:26:00.390 --> 00:26:02.730 ADAM MARTIN: That's possible. 00:26:02.730 --> 00:26:06.180 That's exactly the logic I want to see. 00:26:06.180 --> 00:26:08.488 Is there another possibility? 00:26:08.488 --> 00:26:09.030 Yeah, Jeremy. 00:26:09.030 --> 00:26:10.238 AUDIENCE: Autosomal dominant. 00:26:10.238 --> 00:26:13.380 ADAM MARTIN: It could also be autosomal dominant. 00:26:13.380 --> 00:26:14.488 So you're right. 00:26:14.488 --> 00:26:15.030 You're right. 00:26:15.030 --> 00:26:19.890 If this was not a rare disease, then that male 00:26:19.890 --> 00:26:23.790 could care be a carrier and could be passing it 00:26:23.790 --> 00:26:26.520 on to half the children. 00:26:26.520 --> 00:26:27.720 So that's good. 00:26:27.720 --> 00:26:29.550 You'd essentially need more information 00:26:29.550 --> 00:26:32.760 to differentiate between autosomal recessive 00:26:32.760 --> 00:26:34.620 and autosomal dominant. 00:26:34.620 --> 00:26:36.210 For the purposes of this, we're going 00:26:36.210 --> 00:26:40.500 to go with autosomal dominant. 00:26:40.500 --> 00:26:43.890 And what you see is that you want 00:26:43.890 --> 00:26:46.260 to look at the affected individuals 00:26:46.260 --> 00:26:51.000 and see if the disease phenotype is linked, or connected, 00:26:51.000 --> 00:26:54.480 with one of these microsatellite alleles. 00:26:54.480 --> 00:26:56.530 So if we look at-- 00:26:56.530 --> 00:27:00.750 we basically PCR DNA from all these individuals. 00:27:00.750 --> 00:27:02.700 And if you look at who is affected, 00:27:02.700 --> 00:27:07.500 each one of the individuals has this M double prime band. 00:27:07.500 --> 00:27:12.570 And none of the unaffected individuals has it. 00:27:12.570 --> 00:27:17.970 So obviously, it would be better to have more pedigrees and more 00:27:17.970 --> 00:27:20.370 data to really establish significance 00:27:20.370 --> 00:27:21.870 between this linkage. 00:27:21.870 --> 00:27:24.390 But this is just a simple example, 00:27:24.390 --> 00:27:27.090 showing what you could possibly see 00:27:27.090 --> 00:27:30.120 if you have one of these molecular markers linked 00:27:30.120 --> 00:27:31.860 to a particular disease allele. 00:27:34.770 --> 00:27:38.910 So that kind of establishes the principle. 00:27:38.910 --> 00:27:42.540 Now let's think about what are some other molecular markers 00:27:42.540 --> 00:27:43.290 that are possible? 00:27:47.130 --> 00:27:49.850 So another type of marker, and this 00:27:49.850 --> 00:27:54.210 is one that's the most common one, if I go here. 00:27:54.210 --> 00:27:57.110 So here, you see here's is a linkage map, here. 00:27:57.110 --> 00:28:00.350 And you see most of these bands are green. 00:28:00.350 --> 00:28:03.080 And the green markers, here, are what 00:28:03.080 --> 00:28:07.610 are known as Single Nucleotide Polymorphisms, or SNPs. 00:28:10.420 --> 00:28:14.780 So single nucleotide polymorphisms-- 00:28:24.010 --> 00:28:27.630 and this is abbreviated SNP. 00:28:27.630 --> 00:28:31.730 And what a single nucleotide polymorphism is, is it's 00:28:31.730 --> 00:28:37.680 a variation of a nucleotide at a single position in the genome. 00:28:37.680 --> 00:28:41.880 So it's just a one base pair difference at a position. 00:28:41.880 --> 00:28:54.300 So there's variation of single nucleotide at a given position, 00:28:54.300 --> 00:28:58.380 at a position in the genome. 00:29:02.860 --> 00:29:05.970 And because that's a pretty general definition, 00:29:05.970 --> 00:29:09.780 there are tons of these in the genome. 00:29:09.780 --> 00:29:11.640 Now one thing to think about is you 00:29:11.640 --> 00:29:16.110 could have a mutation in an individual that creates a SNP. 00:29:16.110 --> 00:29:19.710 So you could have a de novo formation of a SNP. 00:29:19.710 --> 00:29:23.580 But if you have a SNP and it gets 00:29:23.580 --> 00:29:26.580 incorporated to the gametes of an individual, 00:29:26.580 --> 00:29:28.710 then that variant is going to be passed 00:29:28.710 --> 00:29:31.260 on to the next generation. 00:29:31.260 --> 00:29:34.110 So this is something that could occur de novo. 00:29:34.110 --> 00:29:36.540 But it is also heritable. 00:29:36.540 --> 00:29:39.810 And if it's heritable, then you can follow it 00:29:39.810 --> 00:29:42.630 and use it to determine if a given 00:29:42.630 --> 00:29:46.155 variant is linked to a given phenotype, like a disease. 00:29:48.730 --> 00:29:53.980 So to identify a single nucleotide polymorphism, 00:29:53.980 --> 00:29:57.190 it's helpful to be able to sequence the DNA. 00:29:57.190 --> 00:30:00.830 And I'll talk about how we could do that in just a minute. 00:30:00.830 --> 00:30:04.060 But before I go on, I just want to point out 00:30:04.060 --> 00:30:10.000 a subclass of SNPs that can be visualized without sequencing. 00:30:10.000 --> 00:30:15.490 And these are called restriction fragment length polymorphisms. 00:30:15.490 --> 00:30:18.640 So restriction fragment-- so it's 00:30:18.640 --> 00:30:20.980 going to involve some type of restriction 00:30:20.980 --> 00:30:26.890 enzyme digest length polymorphism. 00:30:26.890 --> 00:30:28.630 It's a long word. 00:30:28.630 --> 00:30:30.100 But it's abbreviated RFLP. 00:30:32.800 --> 00:30:36.160 And what this is, is it's a variation 00:30:36.160 --> 00:30:37.960 of a single nucleotide. 00:30:37.960 --> 00:30:40.720 But this is a subclass of SNP. 00:30:40.720 --> 00:30:43.810 Because this is when the variation occurs 00:30:43.810 --> 00:30:47.080 in a restriction site for a restriction enzyme. 00:30:47.080 --> 00:30:51.250 So if you remember your good friend EcoR1, 00:30:51.250 --> 00:30:55.270 EcoR1 recognizes the nucleotide sequence GAATTC. 00:30:58.150 --> 00:31:04.270 And EcoR1 only cleaves DNA sequence that has GAATTC. 00:31:07.090 --> 00:31:13.000 So if there was a single nucleotide variation 00:31:13.000 --> 00:31:15.370 in the sequence, such that it's now 00:31:15.370 --> 00:31:24.190 GATTTC, or something like that, that destroys the EcoR1 site. 00:31:24.190 --> 00:31:28.300 And so EcoR1 will no longer be able to recognize this site 00:31:28.300 --> 00:31:31.340 in the genome and cut it. 00:31:31.340 --> 00:31:36.010 So you could imagine that if you had 00:31:36.010 --> 00:31:41.980 one individual in the genome having three EcoR1 sites, 00:31:41.980 --> 00:31:45.700 if you digest this region, you'd get two fragments. 00:31:45.700 --> 00:31:50.720 But if you destroyed the one in the middle, 00:31:50.720 --> 00:31:55.250 then if you digested this piece of DNA, 00:31:55.250 --> 00:31:57.825 then you'd only get one fragment. 00:31:57.825 --> 00:31:58.700 And that's something. 00:31:58.700 --> 00:32:02.000 Because it results in different sizes of fragments, 00:32:02.000 --> 00:32:03.560 that's something you can see just 00:32:03.560 --> 00:32:06.500 by doing DNA electrophoresis. 00:32:06.500 --> 00:32:08.660 And maybe you would use some method 00:32:08.660 --> 00:32:11.600 to detect this specific region, so that you're not 00:32:11.600 --> 00:32:13.820 looking at all the DNA in the genome, 00:32:13.820 --> 00:32:18.080 but you're establishing linkage to this specific area. 00:32:18.080 --> 00:32:20.090 You could use PCR. 00:32:20.090 --> 00:32:23.330 You can have PCR primers here and here. 00:32:23.330 --> 00:32:25.160 And you could then cut with EcoR1. 00:32:25.160 --> 00:32:27.560 In one case, you'd get two fragments. 00:32:27.560 --> 00:32:29.690 In this case, you'd get two fragments. 00:32:29.690 --> 00:32:32.960 In this case, if you amplified this region of the genome 00:32:32.960 --> 00:32:36.140 and cut with EcoR1, you'd only get one fragment. 00:32:36.140 --> 00:32:37.580 So you'd be able to differentiate 00:32:37.580 --> 00:32:39.530 between those possibilities. 00:32:39.530 --> 00:32:40.700 Yes, Malik. 00:32:40.700 --> 00:32:43.490 AUDIENCE: When you use PCR, are there [INAUDIBLE]?? 00:32:45.658 --> 00:32:46.700 ADAM MARTIN: What's that? 00:32:46.700 --> 00:32:48.373 AUDIENCE: Are there [INAUDIBLE]? 00:32:48.373 --> 00:32:49.040 ADAM MARTIN: Oh. 00:32:49.040 --> 00:32:52.500 You're saying what causes it to stop? 00:32:52.500 --> 00:32:53.820 That's a great question, Malik. 00:32:53.820 --> 00:32:54.320 Yeah. 00:32:54.320 --> 00:32:57.620 So initially, it's not going to stop. 00:32:57.620 --> 00:32:59.360 That's absolutely right. 00:32:59.360 --> 00:33:02.540 But because every step, each time you replicate, 00:33:02.540 --> 00:33:06.350 it's then primed with another primer. 00:33:06.350 --> 00:33:10.940 So you'd replicate something like this that's too long. 00:33:10.940 --> 00:33:13.700 But then the reverse primer would replicate like this. 00:33:13.700 --> 00:33:15.650 And it would stop. 00:33:15.650 --> 00:33:19.130 So if you go back to my slide from last lecture, 00:33:19.130 --> 00:33:23.270 look through that and see if it makes sense how it's ending. 00:33:23.270 --> 00:33:25.730 Because if you do this 30 times, you really 00:33:25.730 --> 00:33:30.590 will enrich for a fragment that stops and ends at the two 00:33:30.590 --> 00:33:35.900 primers, or begins and ends at the two primers, I should say. 00:33:35.900 --> 00:33:36.800 Good question. 00:33:36.800 --> 00:33:39.120 Thank you. 00:33:39.120 --> 00:33:39.620 All right. 00:33:39.620 --> 00:33:42.160 Now, let's talk about DNA sequencing. 00:33:42.160 --> 00:33:43.880 Because as I showed you, obviously, 00:33:43.880 --> 00:33:46.280 these SNPs, because there are so many of them, 00:33:46.280 --> 00:33:51.080 are probably the most useful of these markers to narrow in 00:33:51.080 --> 00:33:53.690 on where your disease gene is. 00:33:53.690 --> 00:33:58.160 And to detect a SNP, we need to be able to sequence DNA. 00:34:04.220 --> 00:34:05.810 So I'm going to start with an older 00:34:05.810 --> 00:34:09.530 method for DNA sequencing, which conceptually, 00:34:09.530 --> 00:34:13.250 is very similar to how we do DNA sequencing today. 00:34:13.250 --> 00:34:15.139 And so it will illustrate my point. 00:34:15.139 --> 00:34:19.550 And then at the end, I'll talk about more modern techniques 00:34:19.550 --> 00:34:21.600 to sequencing. 00:34:21.600 --> 00:34:23.570 So the technique I'm going to introduce to you 00:34:23.570 --> 00:34:25.639 is called Sanger sequencing. 00:34:28.820 --> 00:34:33.230 And that's because it was identified by an individual 00:34:33.230 --> 00:34:34.130 named Fred Sanger. 00:34:37.850 --> 00:34:43.429 And I'm going to just take a very simple DNA sequence, 00:34:43.429 --> 00:34:46.600 in order to illustrate how Sanger sequencing works. 00:34:50.370 --> 00:34:53.130 So let's take a sequence that's really simple. 00:35:02.470 --> 00:35:06.810 This is very, very simple, and then more sequence here. 00:35:09.370 --> 00:35:13.800 So let's say we want to determine the nucleotide that's 00:35:13.800 --> 00:35:18.120 at every position of this DNA fragment. 00:35:18.120 --> 00:35:20.790 So one way we could maybe conceptually 00:35:20.790 --> 00:35:22.950 think about doing this, is to try 00:35:22.950 --> 00:35:28.440 to let DNA polymerase tell us where given nucleotides are. 00:35:28.440 --> 00:35:30.690 And if we're going to use DNA polymerase, what 00:35:30.690 --> 00:35:34.780 are we going to need, in order to facilitate this process? 00:35:37.340 --> 00:35:37.970 Yes, Rachel. 00:35:37.970 --> 00:35:39.170 AUDIENCE: [INAUDIBLE]. 00:35:39.170 --> 00:35:42.560 ADAM MARTIN: You're going to need nucleotides, definitely. 00:35:42.560 --> 00:35:44.660 So we're going to need nucleotides. 00:35:44.660 --> 00:35:46.130 What else? 00:35:46.130 --> 00:35:49.080 To start, what are you going to need? 00:35:49.080 --> 00:35:49.916 Miles? 00:35:49.916 --> 00:35:50.770 AUDIENCE: Primer. 00:35:50.770 --> 00:35:52.937 ADAM MARTIN: You're going to need a primer, exactly. 00:35:52.937 --> 00:35:53.920 Good job. 00:35:53.920 --> 00:35:56.680 So you need a primer. 00:35:56.680 --> 00:35:58.660 So here's a primer. 00:35:58.660 --> 00:36:03.130 And now, we're going to try to get DNA polymerase to tell us 00:36:03.130 --> 00:36:06.560 whenever there is a given nucleotide in this DNA 00:36:06.560 --> 00:36:07.060 sequence. 00:36:10.330 --> 00:36:13.540 And so think with me. 00:36:13.540 --> 00:36:19.860 Let's say we were able to get DNA polymerase to stop whenever 00:36:19.860 --> 00:36:22.770 there was a certain nucleotide. 00:36:22.770 --> 00:36:25.980 So if we go through just a couple nucleotides, 00:36:25.980 --> 00:36:32.310 let's say, at first, we want DNA polymerase to stop whenever 00:36:32.310 --> 00:36:37.350 there's an A. So let's say there was 00:36:37.350 --> 00:36:40.400 a possibility it would stop at this A. If it's 00:36:40.400 --> 00:36:42.360 stopped at this A, you'd generate 00:36:42.360 --> 00:36:45.270 a fragment of this length. 00:36:45.270 --> 00:36:47.860 But if it read on through that A, 00:36:47.860 --> 00:36:53.160 there's another possibility that it would stop at this A. 00:36:53.160 --> 00:36:56.730 So we're kind of looking at when these are stopping. 00:36:56.730 --> 00:37:04.200 And the final possibility is it goes on and stops at this A. 00:37:04.200 --> 00:37:07.740 So if this DNA polymerase stopped only at As, 00:37:07.740 --> 00:37:11.580 you'd get fragments that are these three discrete lengths. 00:37:14.440 --> 00:37:17.050 Now let's consider another possibility. 00:37:17.050 --> 00:37:24.160 So pink here is stop at A. And in blue, I'm 00:37:24.160 --> 00:37:27.670 going to draw what would happen if it stopped at T. 00:37:27.670 --> 00:37:30.040 So they all start from the same place. 00:37:30.040 --> 00:37:32.260 If it stopped at T, it would just 00:37:32.260 --> 00:37:36.895 stop one nucleotide beyond this A in this simple sequence. 00:37:39.700 --> 00:37:44.920 So in blue here, this is stop at T. 00:37:44.920 --> 00:37:49.420 But if it's just a possibility, it stops. 00:37:49.420 --> 00:37:53.170 And some of the polymerases could go beyond this T 00:37:53.170 --> 00:37:57.730 and go to the next T and stop here. 00:37:57.730 --> 00:38:01.210 And again, this would be one nucleotide length longer 00:38:01.210 --> 00:38:04.450 than this pink one, here. 00:38:04.450 --> 00:38:06.420 And the final one would-- 00:38:06.420 --> 00:38:07.920 I'll just draw it down here-- 00:38:07.920 --> 00:38:11.380 would get out to this last T, here. 00:38:11.380 --> 00:38:16.130 So what you see is if we could get DNA polymerase to stop 00:38:16.130 --> 00:38:19.480 at these discrete positions, we'd 00:38:19.480 --> 00:38:23.230 get a different sized fragments, whether it 00:38:23.230 --> 00:38:27.730 was stopping at one nucleotide versus the other nucleotide. 00:38:27.730 --> 00:38:30.490 You all see how this is resulting in different fragment 00:38:30.490 --> 00:38:31.780 lengths. 00:38:31.780 --> 00:38:32.820 Yes, Andrew. 00:38:32.820 --> 00:38:35.870 AUDIENCE: How would you create a pattern [INAUDIBLE]?? 00:38:38.710 --> 00:38:40.990 ADAM MARTIN: There are companies now. 00:38:40.990 --> 00:38:42.670 You can basically take nucleotides 00:38:42.670 --> 00:38:46.420 and synthesize these primers chemically, 00:38:46.420 --> 00:38:48.210 not using DNA polymerase. 00:38:48.210 --> 00:38:50.844 AUDIENCE: I'm saying how would you know what primer to use, 00:38:50.844 --> 00:38:52.330 if you don't know the sequence? 00:38:52.330 --> 00:38:53.830 ADAM MARTIN: Oh, in this case, you'd 00:38:53.830 --> 00:38:58.030 have to start with some sequence that you know. 00:38:58.030 --> 00:39:01.150 So in most sequencing technologies, 00:39:01.150 --> 00:39:04.720 you kind of make a DNA library, where you know 00:39:04.720 --> 00:39:06.070 the sequence of the vector. 00:39:06.070 --> 00:39:08.590 And then you'd use the vector sequence as a primer 00:39:08.590 --> 00:39:11.770 to sequence into the unknown sequence. 00:39:11.770 --> 00:39:12.670 Great question. 00:39:12.670 --> 00:39:14.740 Good job. 00:39:14.740 --> 00:39:17.110 All right. 00:39:17.110 --> 00:39:21.190 So what we need now then is some sort of tool or ability 00:39:21.190 --> 00:39:26.080 to stop DNA polymerase when there's a certain nucleotide 00:39:26.080 --> 00:39:28.540 base. 00:39:28.540 --> 00:39:32.200 And to do that, we can use this type of molecule, 00:39:32.200 --> 00:39:36.670 here, which is known as a dideoxynucleotide. 00:39:36.670 --> 00:39:42.070 Remember, for DNA polymerase to elongate a chain, 00:39:42.070 --> 00:39:47.500 it requires that the last base have a three prime hydroxyl. 00:39:47.500 --> 00:39:53.110 And so what this dideoxynucleoside triphosphate 00:39:53.110 --> 00:39:57.040 is, is it's a nucleoside triphosphate that 00:39:57.040 --> 00:39:59.840 lacks a three prime hydroxyl. 00:39:59.840 --> 00:40:02.680 Here, I'll highlight that. 00:40:02.680 --> 00:40:03.820 So you see this guy? 00:40:03.820 --> 00:40:06.150 You see it bolt the highlight H? 00:40:06.150 --> 00:40:09.220 There's a hydrogen there on the three prime carbon, 00:40:09.220 --> 00:40:13.930 rather than the normal hydroxyl group. 00:40:13.930 --> 00:40:20.620 So if this base gets incorporated into a elongating 00:40:20.620 --> 00:40:26.080 chain, DNA polymerase is not going to be able to move on. 00:40:28.690 --> 00:40:34.570 So this method where you can add a certain dideoxynucleoside 00:40:34.570 --> 00:40:38.770 triphosphate to stop chain elongation 00:40:38.770 --> 00:40:42.430 is known as a chain termination method. 00:40:42.430 --> 00:40:46.030 So you're getting chain termination. 00:40:46.030 --> 00:40:48.430 And you're getting this chain termination 00:40:48.430 --> 00:40:52.120 with a specific dideoxynucleoside triphosphate. 00:40:52.120 --> 00:40:56.990 So these dideoxynucleotide triphosphates, 00:40:56.990 --> 00:40:59.770 if they get incorporated into the DNA, 00:40:59.770 --> 00:41:03.590 are going to halt the synthesis of that DNA strand. 00:41:03.590 --> 00:41:08.860 So if we take our example, here, this 00:41:08.860 --> 00:41:14.350 might be a reaction that has dideoxythymidine triphosphate. 00:41:14.350 --> 00:41:21.520 So if we had dideoxythymidine triphosphate in this sample 00:41:21.520 --> 00:41:24.850 and it's elongating, then when the polymerase reaches 00:41:24.850 --> 00:41:26.890 this point, there's a possibility 00:41:26.890 --> 00:41:33.490 that it will incorporate the dideoxynucleoside triphosphate. 00:41:33.490 --> 00:41:37.870 And if this is a dideoxynucleoside triphosphate, 00:41:37.870 --> 00:41:42.070 then there won't be a three prime hydroxyl. 00:41:42.070 --> 00:41:48.340 And DNA polymerase will just be like, oh, I can't go on! 00:41:48.340 --> 00:41:51.420 Because it's not going to have a three prime hydroxyl. 00:41:51.420 --> 00:41:54.010 So it's not going to be able to continue 00:41:54.010 --> 00:41:56.830 with the next nucleotide. 00:41:56.830 --> 00:42:00.040 So this is known as chain termination. 00:42:00.040 --> 00:42:03.415 So let me take you through an example, here. 00:42:06.020 --> 00:42:06.520 All right. 00:42:06.520 --> 00:42:10.330 So here's an example that you have a slide of. 00:42:10.330 --> 00:42:13.520 And again, there's a template strand, 00:42:13.520 --> 00:42:15.520 which is the top strand. 00:42:15.520 --> 00:42:19.330 And this method requires that you have a primer. 00:42:19.330 --> 00:42:21.535 And what's often done is you label the primer. 00:42:24.790 --> 00:42:28.690 So the first step is you have to denature your DNA. 00:42:28.690 --> 00:42:30.820 So you have to go from double stranded DNA 00:42:30.820 --> 00:42:32.050 to a single stranded DNA. 00:42:34.730 --> 00:42:41.000 And then you mix the double stranded DNA 00:42:41.000 --> 00:42:45.110 with first, this labeled primer, such 00:42:45.110 --> 00:42:49.940 that the primer can then yield to the single stranded DNA. 00:42:49.940 --> 00:42:53.846 You need DNA polymerase, as I've mentioned. 00:42:57.740 --> 00:42:59.990 And as, I believe, Rachel mentioned 00:42:59.990 --> 00:43:03.650 before, you need the building blocks of DNA. 00:43:03.650 --> 00:43:06.680 So you need the four dideoxynucleoside 00:43:06.680 --> 00:43:08.450 triphosphates. 00:43:08.450 --> 00:43:11.330 So you always have the four dideoxynucleotide 00:43:11.330 --> 00:43:14.030 triphosphates. 00:43:14.030 --> 00:43:16.100 But what's special here is you're 00:43:16.100 --> 00:43:20.480 going to spike several reactions with one 00:43:20.480 --> 00:43:23.630 of the dideoxynucleoside triphosphates. 00:43:23.630 --> 00:43:28.400 So you spike the reaction with a tiny amount 00:43:28.400 --> 00:43:34.190 of one of your dideoxynucleoside triphosphates. 00:43:36.860 --> 00:43:39.770 So let's say you have a reaction, here. 00:43:39.770 --> 00:43:45.970 And this this one here has dideoxyadenosine triphosphate. 00:43:45.970 --> 00:43:48.800 Then polymerase will along get this strand 00:43:48.800 --> 00:43:52.040 until there's a thymidine on the template. 00:43:52.040 --> 00:43:53.870 And then there's a possibility that it will 00:43:53.870 --> 00:43:57.170 incorporate this dideoxy NTP. 00:43:57.170 --> 00:43:59.480 And if it does, then you get chain termination. 00:43:59.480 --> 00:44:01.760 And you get a fragment of this length. 00:44:01.760 --> 00:44:04.460 But the other possibility, because there is still 00:44:04.460 --> 00:44:08.420 the deoxy form of the NTP present, 00:44:08.420 --> 00:44:10.550 it's possible that it incorporates 00:44:10.550 --> 00:44:13.760 a deoxyadenosine triphosphate there. 00:44:13.760 --> 00:44:18.050 And keeps going, and then incorporates a dideoxy ATP 00:44:18.050 --> 00:44:21.890 later on, where you have another T. 00:44:21.890 --> 00:44:24.800 And so the polymerase will essentially randomly 00:44:24.800 --> 00:44:30.410 stop at these different thymidine residues, 00:44:30.410 --> 00:44:34.070 depending on whether or not a dideoxynucleoside triphosphate 00:44:34.070 --> 00:44:35.690 is incorporated. 00:44:35.690 --> 00:44:38.480 And that means for a given reaction, one in which 00:44:38.480 --> 00:44:42.230 you have dideoxy ATP, you get a certain pattern 00:44:42.230 --> 00:44:46.640 of bands that represent the length of fragments, 00:44:46.640 --> 00:44:52.130 where you have, in this case, a thymidine base. 00:44:52.130 --> 00:44:55.790 And then you do this for all four bases, where 00:44:55.790 --> 00:44:59.410 you have four reactions, each with a different base that's 00:44:59.410 --> 00:44:59.910 dideoxy. 00:45:02.510 --> 00:45:04.700 So when you're adding these, you're 00:45:04.700 --> 00:45:10.820 going to do four reactions, one with dideoxy ATP spiked in, 00:45:10.820 --> 00:45:15.860 one with dideoxy TTP, one with dideoxy CTP, 00:45:15.860 --> 00:45:19.280 and the last with dideoxy GTP. 00:45:19.280 --> 00:45:22.280 And because these nucleotides are 00:45:22.280 --> 00:45:26.360 present in different positions along the sequence, 00:45:26.360 --> 00:45:30.080 you're going to get distinct banding pattern for each 00:45:30.080 --> 00:45:31.700 of these reactions. 00:45:31.700 --> 00:45:33.500 But using that banding pattern, you 00:45:33.500 --> 00:45:36.200 can then read off the sequence of DNA that's 00:45:36.200 --> 00:45:37.850 present on the template strand. 00:45:41.180 --> 00:45:45.590 So this is how sequencing was done for many, many years. 00:45:45.590 --> 00:45:50.030 These days, it's been made cheaper and faster. 00:45:50.030 --> 00:45:54.750 And now what's often used is next generation sequencing. 00:45:54.750 --> 00:45:59.090 And one the pain in the ass about sequencing before 00:45:59.090 --> 00:46:02.000 is you'd use a lot of radioactivity. 00:46:02.000 --> 00:46:05.020 Your primer would be radioactive, 00:46:05.020 --> 00:46:07.280 so that you could detect these bands. 00:46:07.280 --> 00:46:09.680 Right now, everything's done using fluorescence, which 00:46:09.680 --> 00:46:11.810 makes it much nicer, I think. 00:46:11.810 --> 00:46:13.940 And so in next generation sequencing, 00:46:13.940 --> 00:46:18.140 your template DNA is attached to a solid substrate, such 00:46:18.140 --> 00:46:22.880 that it's immobilized on some type of substrate. 00:46:22.880 --> 00:46:29.840 And then you add each of the four nucleoside triphosphates. 00:46:29.840 --> 00:46:33.290 In this case, they're labeled with a dye, such 00:46:33.290 --> 00:46:35.660 that each one is a different color. 00:46:35.660 --> 00:46:39.530 But the dye also functions to prevent elongation, 00:46:39.530 --> 00:46:42.500 such that, again, it's this chain termination. 00:46:42.500 --> 00:46:44.330 When you incorporate one of these, 00:46:44.330 --> 00:46:47.150 the polymerase just can't run along the DNA. 00:46:47.150 --> 00:46:50.300 It incorporates one and then stops. 00:46:50.300 --> 00:46:53.720 So if you get your first nucleotide incorporated, 00:46:53.720 --> 00:46:56.060 it will incorporate one of these four. 00:46:56.060 --> 00:46:59.330 And it will be fluorescent at a certain wavelength, which 00:46:59.330 --> 00:47:04.880 you can see using a device or microscope. 00:47:04.880 --> 00:47:08.900 And then what you then do is chemically modify this base, 00:47:08.900 --> 00:47:11.300 such that you remove the dye and allow it 00:47:11.300 --> 00:47:13.910 to extend one more base pair. 00:47:13.910 --> 00:47:17.490 And so you go one nucleotide at a time. 00:47:17.490 --> 00:47:20.480 And you read out the pattern of fluorescence that appears. 00:47:20.480 --> 00:47:23.450 And that gives you the sequence of DNA 00:47:23.450 --> 00:47:27.230 on this molecule that's stuck to your substrate. 00:47:27.230 --> 00:47:28.580 And you can do this in parallel. 00:47:28.580 --> 00:47:32.120 You can have tons, many different strands of DNA. 00:47:32.120 --> 00:47:34.430 And you can be reading out the sequence of each one 00:47:34.430 --> 00:47:36.200 of these strands in parallel. 00:47:39.060 --> 00:47:39.560 Great. 00:47:39.560 --> 00:47:41.480 Any questions about DNA sequencing? 00:47:45.200 --> 00:47:45.700 OK. 00:47:45.700 --> 00:47:46.420 Very good. 00:47:46.420 --> 00:47:47.630 I will see you on Monday. 00:47:47.630 --> 00:47:49.620 Have a great weekend.