1 00:00:16,490 --> 00:00:19,170 PROFESSOR: All right, so fluorescence. 2 00:00:19,170 --> 00:00:21,270 You know, I know you hear this from me a lot. 3 00:00:21,270 --> 00:00:24,570 But this really is my favorite topic. 4 00:00:24,570 --> 00:00:27,990 The applications of luminescence and fluorescence 5 00:00:27,990 --> 00:00:32,650 in service to biology are incredibly important. 6 00:00:32,650 --> 00:00:35,820 So what I'm going to try to do in these two lectures 7 00:00:35,820 --> 00:00:39,570 is explain to you the difference between fluorophores 8 00:00:39,570 --> 00:00:44,220 that we can encode into proteins through genetic engineering 9 00:00:44,220 --> 00:00:46,440 and fluorophores that we use that are 10 00:00:46,440 --> 00:00:50,340 made by chemists in the lab but then appended to molecules. 11 00:00:50,340 --> 00:00:54,480 So today we'll talk about the nuts and bolts of fluorescence. 12 00:00:54,480 --> 00:00:56,100 And then on Wednesday, we'll start 13 00:00:56,100 --> 00:01:01,740 to see some of these tools that you've seen images of. 14 00:01:01,740 --> 00:01:05,640 We love to wow you with images of fluorescent cells and cells 15 00:01:05,640 --> 00:01:06,480 in action. 16 00:01:06,480 --> 00:01:09,510 But I want to step back and actually show you how 17 00:01:09,510 --> 00:01:11,280 that all came about. 18 00:01:11,280 --> 00:01:13,900 Where do these fluorescent proteins come from? 19 00:01:13,900 --> 00:01:15,300 What are we looking for? 20 00:01:15,300 --> 00:01:18,090 How much protein engineering was done 21 00:01:18,090 --> 00:01:22,470 to make these such an amazingly useful set of molecules, 22 00:01:22,470 --> 00:01:26,280 macromolecules to really allow us in real time 23 00:01:26,280 --> 00:01:28,180 to study biology? 24 00:01:28,180 --> 00:01:31,630 And there are many, many other applications as well. 25 00:01:31,630 --> 00:01:35,130 So we're going to talk about luminescence and fluorescence 26 00:01:35,130 --> 00:01:36,450 in general. 27 00:01:36,450 --> 00:01:38,415 Luminescence is the general term. 28 00:01:43,830 --> 00:01:47,350 And fluorescence is a little bit more specific. 29 00:01:47,350 --> 00:01:49,660 There are different types of luminescence. 30 00:01:49,660 --> 00:01:51,910 And you'll get to see some of those varieties 31 00:01:51,910 --> 00:01:53,080 of luminescence. 32 00:01:53,080 --> 00:01:56,660 I've put a decent amount of our content today on the screen. 33 00:01:56,660 --> 00:01:58,580 So we'll go up here and take a look. 34 00:01:58,580 --> 00:02:02,020 So luminescence in general is the emission 35 00:02:02,020 --> 00:02:05,290 of light not associated with heat, not 36 00:02:05,290 --> 00:02:09,190 like a burning flame which has a lot of light accompanying it. 37 00:02:09,190 --> 00:02:13,500 But rather the emission of light in the absence of heat. 38 00:02:13,500 --> 00:02:18,580 And there are different types of luminescence the biologists use 39 00:02:18,580 --> 00:02:21,850 intertwined into biological experiments 40 00:02:21,850 --> 00:02:24,100 to illuminate life, to understand 41 00:02:24,100 --> 00:02:26,710 details of cellular activity. 42 00:02:26,710 --> 00:02:30,040 But also as reagents in diagnostics 43 00:02:30,040 --> 00:02:33,340 in all kinds of imaging modalities. 44 00:02:33,340 --> 00:02:34,600 And through these two-- 45 00:02:34,600 --> 00:02:37,270 three lectures, actually, because Professor Martin 46 00:02:37,270 --> 00:02:39,970 will give you one that has even more imaging in it-- 47 00:02:39,970 --> 00:02:43,630 you'll sort of really get to understand where these pretty 48 00:02:43,630 --> 00:02:45,950 magical reagents come from. 49 00:02:45,950 --> 00:02:48,100 So the two types of luminescence that we 50 00:02:48,100 --> 00:02:53,200 won't discuss in detail today, first of all chemiluminescence. 51 00:02:53,200 --> 00:02:56,140 This is a molecule known as luminol 52 00:02:56,140 --> 00:02:57,610 sitting in a little bile. 53 00:02:57,610 --> 00:03:01,210 I think I like fluorescence so much because the images are so 54 00:03:01,210 --> 00:03:02,650 captivating. 55 00:03:02,650 --> 00:03:05,890 Now, things like luminol, has anyone heard of luminol before? 56 00:03:05,890 --> 00:03:09,430 Does anyone-- yeah, do you watch a lot of CSI or-- 57 00:03:09,430 --> 00:03:10,090 yeah. 58 00:03:10,090 --> 00:03:13,935 So tell everybody what people use luminol for. 59 00:03:13,935 --> 00:03:16,590 AUDIENCE: To pick up on a blood spatter or remnants of blood. 60 00:03:16,590 --> 00:03:20,170 PROFESSOR: Yeah, so when you see these TV 61 00:03:20,170 --> 00:03:24,580 shows and there's this beautifully clean motel room 62 00:03:24,580 --> 00:03:27,880 and nothing looks like it ever happened there. 63 00:03:27,880 --> 00:03:30,880 But people thought there was a murder took place there, 64 00:03:30,880 --> 00:03:33,790 you'll notice they come in with these spray bottles. 65 00:03:33,790 --> 00:03:37,623 And spray all over the carpets, and the drapes, and the chairs. 66 00:03:37,623 --> 00:03:39,040 And then there's this great moment 67 00:03:39,040 --> 00:03:43,420 where they turn off the light and luminol interacts 68 00:03:43,420 --> 00:03:48,280 with the heme of blood at an amazingly sensitive level, 69 00:03:48,280 --> 00:03:50,650 such that when the lights are turned out, 70 00:03:50,650 --> 00:03:53,950 the room's just sort of this battlefield 71 00:03:53,950 --> 00:03:56,560 of bright luminescence that indicates 72 00:03:56,560 --> 00:03:58,130 that this was a crime scene. 73 00:03:58,130 --> 00:04:03,180 So that's the most famous luminol sort of example. 74 00:04:03,180 --> 00:04:05,690 So that is what would be called chemiluminescence, 75 00:04:05,690 --> 00:04:08,650 the interaction of a chemical with another chemical 76 00:04:08,650 --> 00:04:10,420 to give luminescence. 77 00:04:10,420 --> 00:04:12,900 Another pretty useful type of luminescence 78 00:04:12,900 --> 00:04:14,890 is bioluminescence. 79 00:04:14,890 --> 00:04:16,959 I've done a lot of scuba diving in my life. 80 00:04:16,959 --> 00:04:20,290 And there's nothing more exciting than a dive at night 81 00:04:20,290 --> 00:04:24,220 where the whole ocean is this sort of inky black. 82 00:04:24,220 --> 00:04:26,080 And sometimes you'll move your arms 83 00:04:26,080 --> 00:04:28,450 through the black water at night. 84 00:04:28,450 --> 00:04:31,470 And you'll see all these, like, little fireworks. 85 00:04:31,470 --> 00:04:35,530 And many, many marine organisms actually 86 00:04:35,530 --> 00:04:37,600 undergo bioluminescence. 87 00:04:37,600 --> 00:04:41,170 It's a biological reaction that causes luminescence. 88 00:04:41,170 --> 00:04:44,110 And this is a cuttlefish shown here 89 00:04:44,110 --> 00:04:47,560 in this image in the corner where 90 00:04:47,560 --> 00:04:49,550 they are brightly lit at night. 91 00:04:49,550 --> 00:04:51,340 And actually, it's just a whole party 92 00:04:51,340 --> 00:04:54,820 there at night in the ocean where all sorts of organisms 93 00:04:54,820 --> 00:04:58,870 are signaling to other organisms through bioluminescence 94 00:04:58,870 --> 00:05:01,850 and reactions such as luciferase reactions. 95 00:05:01,850 --> 00:05:04,870 So in bioluminescence, a molecule of ATP 96 00:05:04,870 --> 00:05:09,130 is generally used and combined with another molecule 97 00:05:09,130 --> 00:05:11,680 through the action of an enzyme that ends up 98 00:05:11,680 --> 00:05:13,700 kicking out light energy. 99 00:05:13,700 --> 00:05:14,960 So those are both important. 100 00:05:14,960 --> 00:05:17,740 But what we're going to talk about principally 101 00:05:17,740 --> 00:05:19,690 is fluorescence. 102 00:05:19,690 --> 00:05:21,455 This is a more specific term. 103 00:05:26,370 --> 00:05:28,180 And you may wonder why I'm putting 104 00:05:28,180 --> 00:05:31,158 this in capital letters. 105 00:05:31,158 --> 00:05:32,950 The first thing to learn about fluorescence 106 00:05:32,950 --> 00:05:35,560 is how to spell fluorescence. 107 00:05:35,560 --> 00:05:37,480 So if you look at the word fluorescence 108 00:05:37,480 --> 00:05:41,950 and the first word first part of the word looks like flower, 109 00:05:41,950 --> 00:05:44,620 you know the stuff you bake your pumpkin pie with, 110 00:05:44,620 --> 00:05:46,270 you spelled it wrong. 111 00:05:46,270 --> 00:05:50,722 It actually is fluor, F-L-U-O-R-E-S. 112 00:05:50,722 --> 00:05:52,180 What are you guys whispering about? 113 00:05:52,180 --> 00:05:53,530 AUDIENCE: [INAUDIBLE]. 114 00:05:53,530 --> 00:05:55,280 PROFESSOR: Did I get something else wrong? 115 00:05:55,280 --> 00:05:56,471 AUDIENCE: The E-S. 116 00:05:56,471 --> 00:05:59,420 PROFESSOR: E-S, yeah, well, forget about that part. 117 00:05:59,420 --> 00:06:02,550 E-S. There's another C in there. 118 00:06:02,550 --> 00:06:04,520 There we go, we snuggled that in. 119 00:06:04,520 --> 00:06:07,140 So the important part is the first part. 120 00:06:07,140 --> 00:06:09,770 So make it look like you know what you're talking about 121 00:06:09,770 --> 00:06:11,390 and spell fluorescence correctly. 122 00:06:11,390 --> 00:06:14,810 I cannot tell you how many papers, 123 00:06:14,810 --> 00:06:17,340 scientific papers I read where they spelled fluorescence 124 00:06:17,340 --> 00:06:17,840 wrong. 125 00:06:17,840 --> 00:06:19,340 It's really hysterical. 126 00:06:19,340 --> 00:06:20,240 It's one of those-- 127 00:06:20,240 --> 00:06:23,300 there's two or three amazingly common typos 128 00:06:23,300 --> 00:06:24,440 in people's slides. 129 00:06:24,440 --> 00:06:26,750 One of them is spelling fluorescence wrong 130 00:06:26,750 --> 00:06:29,870 and the other one is spelling complement wrong. 131 00:06:29,870 --> 00:06:31,610 Complement as opposed to compliment 132 00:06:31,610 --> 00:06:33,530 where you're telling someone they look good. 133 00:06:33,530 --> 00:06:34,970 And complement where you're trying 134 00:06:34,970 --> 00:06:37,710 to sort of match up things. 135 00:06:37,710 --> 00:06:40,250 Anyway, but fluorescence is a key point. 136 00:06:40,250 --> 00:06:41,870 So what is fluorescence? 137 00:06:41,870 --> 00:06:58,030 It's the absorption of light energy by a molecule. 138 00:06:58,030 --> 00:07:01,330 Now it could be a small, organic molecule. 139 00:07:01,330 --> 00:07:05,020 It could be a small part of a fluorescent protein 140 00:07:05,020 --> 00:07:07,450 molecule that has a particular structure. 141 00:07:07,450 --> 00:07:11,350 But it will absorb light energy at a certain wavelength. 142 00:07:11,350 --> 00:07:15,070 So let's put this into just a cuvette experiment. 143 00:07:15,070 --> 00:07:19,090 These are the kinds of little containers 144 00:07:19,090 --> 00:07:21,470 that we use for certain types of fluorescence. 145 00:07:21,470 --> 00:07:24,220 We could use a plate and a plate reader. 146 00:07:24,220 --> 00:07:25,840 But this is quite common. 147 00:07:25,840 --> 00:07:28,170 So you shine light on this molecule. 148 00:07:32,090 --> 00:07:35,540 Do you want to nab the doors, the outer doors on both sides? 149 00:07:35,540 --> 00:07:36,150 I don't know. 150 00:07:36,150 --> 00:07:38,080 Everybody's pretty happy today. 151 00:07:38,080 --> 00:07:40,590 Anyway, and the molecule goes. 152 00:07:40,590 --> 00:07:45,430 And I'm going to just draw something, 153 00:07:45,430 --> 00:07:49,620 you know, with a bunch of double bonds and things. 154 00:07:49,620 --> 00:07:54,700 And the molecule absorbs light and goes to an excited state. 155 00:07:54,700 --> 00:08:00,300 So this is the ground state before light. 156 00:08:00,300 --> 00:08:09,070 After light you get the molecule in an excited state. 157 00:08:09,070 --> 00:08:13,540 So it has absorbed that light energy. 158 00:08:13,540 --> 00:08:16,980 So you've hit it with a wavelength of light. 159 00:08:16,980 --> 00:08:19,590 And I'm going to redefine all these terms properly 160 00:08:19,590 --> 00:08:20,700 in a moment. 161 00:08:20,700 --> 00:08:26,250 Lambda excitation of a particular wavelength. 162 00:08:26,250 --> 00:08:29,010 Once that molecule has absorbed light, 163 00:08:29,010 --> 00:08:33,750 there's a very transient period until the molecule lets out 164 00:08:33,750 --> 00:08:35,205 energy in the form of light. 165 00:08:42,669 --> 00:08:46,090 And returns back to its ground state now. 166 00:08:49,510 --> 00:08:52,510 So that the photo physics of fluorescence 167 00:08:52,510 --> 00:08:55,510 involves the excitation of a molecule with light 168 00:08:55,510 --> 00:08:56,980 of one energy. 169 00:08:56,980 --> 00:08:59,670 That light energy is at a particular wavelength 170 00:08:59,670 --> 00:09:03,910 so it's called its lambda of excitation. 171 00:09:03,910 --> 00:09:06,130 That molecule very, very transiently, 172 00:09:06,130 --> 00:09:08,620 it's usually picoseconds or nanoseconds 173 00:09:08,620 --> 00:09:10,240 for organic molecules. 174 00:09:10,240 --> 00:09:13,870 It may be a little bit longer for other types of complexes 175 00:09:13,870 --> 00:09:17,050 and may stretch to the microsecond or millisecond. 176 00:09:17,050 --> 00:09:19,750 But most of what we talk about will be picosecond 177 00:09:19,750 --> 00:09:21,730 to nanosecond lifetime. 178 00:09:21,730 --> 00:09:25,810 And once it's excited, it just drops its energy back out 179 00:09:25,810 --> 00:09:27,670 and goes back to the ground state. 180 00:09:27,670 --> 00:09:31,030 And the most important thing that you want to remember 181 00:09:31,030 --> 00:09:41,510 is the wavelength, which is given in nanometers. 182 00:09:41,510 --> 00:09:49,000 And the wavelength for emission, which is also in nanometers. 183 00:09:49,000 --> 00:09:51,200 This wavelength is higher energy. 184 00:09:55,110 --> 00:09:57,520 Obviously you don't create energy 185 00:09:57,520 --> 00:09:59,560 when you shine light on something. 186 00:09:59,560 --> 00:10:02,650 You'd be breaking a few fundamental rules if you did. 187 00:10:02,650 --> 00:10:07,870 So the light that comes back out is lower energy 188 00:10:07,870 --> 00:10:10,570 because there's been rearrangements in the excited 189 00:10:10,570 --> 00:10:11,750 state of the molecule. 190 00:10:11,750 --> 00:10:16,420 So you can't possibly kick energy out at a higher energy. 191 00:10:16,420 --> 00:10:24,700 And so this is a shorter wavelength in nanometers. 192 00:10:24,700 --> 00:10:26,926 And this is at a longer wavelength. 193 00:10:31,180 --> 00:10:34,640 So remember, higher energy are shorter wavelength. 194 00:10:34,640 --> 00:10:37,340 Lower energy are longer wavelengths. 195 00:10:37,340 --> 00:10:39,650 And that is a rule for fluorescence. 196 00:10:39,650 --> 00:10:41,510 When you excite a molecule, you'll 197 00:10:41,510 --> 00:10:43,430 take it to the excited state. 198 00:10:43,430 --> 00:10:45,680 It'll sit and vibrate there a little bit. 199 00:10:45,680 --> 00:10:48,590 Then it will kick back energy out at a longer wavelength. 200 00:10:48,590 --> 00:10:52,220 And for the majority of the fluorescence experiments 201 00:10:52,220 --> 00:10:55,070 that we do in biology, the wavelengths 202 00:10:55,070 --> 00:11:03,020 that you see emission at are in the visible range. 203 00:11:03,020 --> 00:11:07,280 Whereas the wavelengths that you might excite your molecule at 204 00:11:07,280 --> 00:11:16,740 are often in the UV or a bit longer, ideally, longer. 205 00:11:16,740 --> 00:11:18,597 And these things are going to come up. 206 00:11:18,597 --> 00:11:20,930 This isn't the first time that you're going to see them. 207 00:11:20,930 --> 00:11:23,710 And it's not the last time you're going to see them. 208 00:11:23,710 --> 00:11:26,270 So let's take a look at fluorescent dyes 209 00:11:26,270 --> 00:11:29,940 in the electromagnetic spectrum in the next couple of slides. 210 00:11:29,940 --> 00:11:39,580 So what you see here, you see a bunch of little eppendorf vials 211 00:11:39,580 --> 00:11:42,700 with fluorescent molecules that emit light 212 00:11:42,700 --> 00:11:44,680 at all different wavelengths. 213 00:11:44,680 --> 00:11:47,110 These would be down at the ultraviolet end 214 00:11:47,110 --> 00:11:49,480 of the electromagnetic spectrum. 215 00:11:49,480 --> 00:11:53,920 These would be up in the very red end, the lowest energy. 216 00:11:53,920 --> 00:11:56,560 So these emission wavelengths, these 217 00:11:56,560 --> 00:12:00,880 would be emitting at the lowest energy, longest wavelength. 218 00:12:00,880 --> 00:12:04,780 These would be emitting at the shortest wavelength, lowest 219 00:12:04,780 --> 00:12:06,268 to highest energy. 220 00:12:06,268 --> 00:12:07,310 Is everyone following me? 221 00:12:07,310 --> 00:12:09,180 So just make sure you remember that. 222 00:12:09,180 --> 00:12:12,490 And just this principle rule that you can't possibly 223 00:12:12,490 --> 00:12:15,160 break with respect to the wavelengths of light 224 00:12:15,160 --> 00:12:17,290 for fluorescence experiments. 225 00:12:17,290 --> 00:12:20,710 So what we're going to see is the relationship 226 00:12:20,710 --> 00:12:22,140 for the electromagnetic spectrum. 227 00:12:22,140 --> 00:12:24,130 There's a little bit more detail in a minute. 228 00:12:24,130 --> 00:12:26,110 And then look at some fluorescent dyes 229 00:12:26,110 --> 00:12:29,240 that are very, very commonly used in biology. 230 00:12:29,240 --> 00:12:31,690 And in fact, you will have seen a lot 231 00:12:31,690 --> 00:12:35,500 of cells stained with these dyes even so far in pictures 232 00:12:35,500 --> 00:12:37,300 that you've seen on the screen. 233 00:12:37,300 --> 00:12:41,020 And then we'll talk about the application of antibody 234 00:12:41,020 --> 00:12:43,300 reagents and where does that come in with respect 235 00:12:43,300 --> 00:12:44,590 to fluorescences. 236 00:12:44,590 --> 00:12:46,570 So let's take a look at fluorescence 237 00:12:46,570 --> 00:12:48,520 and the electromagnetic spectrum. 238 00:12:48,520 --> 00:12:52,720 So here it is, going from wavelengths. 239 00:12:52,720 --> 00:12:55,870 The ultraviolet wavelengths would be shorter 240 00:12:55,870 --> 00:12:58,420 than 400 nanometers. 241 00:12:58,420 --> 00:13:01,300 So ultraviolet, so beyond violet. 242 00:13:01,300 --> 00:13:05,230 And the very red wavelengths would be in the range 243 00:13:05,230 --> 00:13:07,330 from 600 to 700. 244 00:13:07,330 --> 00:13:09,580 And here you see the relationship 245 00:13:09,580 --> 00:13:15,640 between wavelength, and then what the light emitted 246 00:13:15,640 --> 00:13:16,610 would look like. 247 00:13:16,610 --> 00:13:20,230 So if we're looking at here, what we would expect to see 248 00:13:20,230 --> 00:13:23,930 is if we've got a fluorophore and it shows fluorescence, 249 00:13:23,930 --> 00:13:26,530 we would be exciting the fluorophore 250 00:13:26,530 --> 00:13:28,530 in wavelengths in this region. 251 00:13:28,530 --> 00:13:31,510 And we would see emission in wavelengths in this region. 252 00:13:31,510 --> 00:13:36,040 But a cardinal rule is that we excite with a shorter, higher 253 00:13:36,040 --> 00:13:38,950 energy wavelength and observe emission 254 00:13:38,950 --> 00:13:42,020 at a longer, lower energy wavelength. 255 00:13:42,020 --> 00:13:45,220 Now, there's very important dyes. 256 00:13:45,220 --> 00:13:47,500 Straight away the most common dye 257 00:13:47,500 --> 00:13:50,800 that you will see immediately in biology 258 00:13:50,800 --> 00:13:52,900 is a dye known as ethidium bromide. 259 00:13:52,900 --> 00:13:54,640 And here's its structure. 260 00:13:54,640 --> 00:13:57,190 You can often recognize fluorophores. 261 00:13:57,190 --> 00:14:00,640 They have lots of rings fused together with lots 262 00:14:00,640 --> 00:14:02,450 of double bonds in them. 263 00:14:02,450 --> 00:14:04,240 This is the structure of a compound 264 00:14:04,240 --> 00:14:06,610 known as ethidium bromide. 265 00:14:06,610 --> 00:14:11,480 It's a dye that intercalates into DNA. 266 00:14:11,480 --> 00:14:13,900 And when the dye changes its environment 267 00:14:13,900 --> 00:14:19,390 from being in water to being snuggled in between stacks 268 00:14:19,390 --> 00:14:23,920 of base pairs in DNA, it changes its fluorescent properties 269 00:14:23,920 --> 00:14:26,020 and it becomes fluorescence. 270 00:14:26,020 --> 00:14:30,290 So fluorescence isn't just the intrinsic shape of the molecule 271 00:14:30,290 --> 00:14:31,630 and what it looks like. 272 00:14:31,630 --> 00:14:35,980 It's very, very often related to what's around it. 273 00:14:35,980 --> 00:14:37,520 Why is that the case? 274 00:14:37,520 --> 00:14:41,680 It's because the excited state may behave differently 275 00:14:41,680 --> 00:14:43,420 in different environments. 276 00:14:43,420 --> 00:14:45,940 Maybe stabilize for a while, and that's 277 00:14:45,940 --> 00:14:49,330 why you might see fluorophores experience 278 00:14:49,330 --> 00:14:51,910 a change in their fluorescence as a function 279 00:14:51,910 --> 00:14:53,100 of their environment. 280 00:14:53,100 --> 00:14:54,580 Is that clear to everyone? 281 00:14:54,580 --> 00:14:57,550 So the molecular environments, if I'm a fluorophore 282 00:14:57,550 --> 00:15:00,220 and I'm in water, I'm going to feel pretty differently 283 00:15:00,220 --> 00:15:02,620 in my excited state if I'm a fluorophore 284 00:15:02,620 --> 00:15:06,100 and I'm sitting packed between DNA bases. 285 00:15:06,100 --> 00:15:08,230 It's pretty dramatic when you see it. 286 00:15:08,230 --> 00:15:11,590 So when you mix ethidium bromide with DNA, 287 00:15:11,590 --> 00:15:15,550 and it could be in a cell or it could be a lysate from a cell 288 00:15:15,550 --> 00:15:19,120 where you're capturing the DNA and trying to manipulate it 289 00:15:19,120 --> 00:15:21,130 in recombinant biology. 290 00:15:21,130 --> 00:15:25,180 That ethidium bromide will intercalate into the DNA. 291 00:15:25,180 --> 00:15:28,840 And it will light up as a bright orange dye. 292 00:15:28,840 --> 00:15:34,330 So here, say we've got a gel that we've run DNA on. 293 00:15:34,330 --> 00:15:36,850 We might have a set of standards. 294 00:15:36,850 --> 00:15:40,690 And in other places, we're looking for the size of DNA. 295 00:15:40,690 --> 00:15:42,430 Remember that great experiment we 296 00:15:42,430 --> 00:15:46,720 saw where we saw how quickly small and large pieces of DNA 297 00:15:46,720 --> 00:15:48,910 ran through an agarose gel? 298 00:15:48,910 --> 00:15:52,030 So here is what the DNA gel would 299 00:15:52,030 --> 00:15:56,110 look like if you soaked it ethidium bromide. 300 00:15:56,110 --> 00:15:58,600 So here's the gel as a ladder of bands. 301 00:15:58,600 --> 00:16:02,950 But then let's say you wanted to do some work on a piece of DNA 302 00:16:02,950 --> 00:16:07,960 and maybe ligated you see DNA pieces at different wavelengths 303 00:16:07,960 --> 00:16:11,080 that have different mobilities based on size. 304 00:16:11,080 --> 00:16:14,202 We couldn't see the DNA directly. 305 00:16:14,202 --> 00:16:15,160 We couldn't pick it up. 306 00:16:15,160 --> 00:16:16,600 We couldn't visualize it. 307 00:16:16,600 --> 00:16:19,190 So we have to use ways to visualize it. 308 00:16:19,190 --> 00:16:21,630 One way is to radio label it. 309 00:16:21,630 --> 00:16:23,830 Messy, we don't really want to do a lot of that 310 00:16:23,830 --> 00:16:25,060 if we can avoid it. 311 00:16:25,060 --> 00:16:28,450 The other way is simply to soak a dye into the gel. 312 00:16:28,450 --> 00:16:31,780 And the dye, because of the positive charge here, 313 00:16:31,780 --> 00:16:33,790 the counter ion gets displaced. 314 00:16:33,790 --> 00:16:36,130 And the positive charge gets attracted 315 00:16:36,130 --> 00:16:39,970 to the DNA and associates with it quite tightly. 316 00:16:39,970 --> 00:16:43,810 So that would be a way that you would observe 317 00:16:43,810 --> 00:16:47,200 DNA bound to dye in a gel. 318 00:16:47,200 --> 00:16:50,270 And this fluoresces a pretty long wavelength. 319 00:16:50,270 --> 00:16:52,690 So this fluoresces this bright orange 320 00:16:52,690 --> 00:16:54,970 that's actually at about 605. 321 00:16:54,970 --> 00:16:57,490 So you can see, this is really in the visible range. 322 00:16:57,490 --> 00:16:59,770 605 would be right around here. 323 00:16:59,770 --> 00:17:02,950 And it has this bright kind of orange fluorescence. 324 00:17:02,950 --> 00:17:04,329 And the wavelength that you would 325 00:17:04,329 --> 00:17:07,390 use to irradiate the dye on the DNA 326 00:17:07,390 --> 00:17:10,390 would be a shorter wavelength than 605. 327 00:17:10,390 --> 00:17:12,099 You will often have a prescription, 328 00:17:12,099 --> 00:17:15,970 excite at this wavelength so you observe at this wavelength. 329 00:17:15,970 --> 00:17:18,770 And these are fixed physical parameters 330 00:17:18,770 --> 00:17:21,550 for fluorescent molecules. 331 00:17:21,550 --> 00:17:27,790 Now, ethidium bromide is a dye that can get into cells. 332 00:17:27,790 --> 00:17:30,790 And we can look at DNA within cells. 333 00:17:30,790 --> 00:17:33,100 And here's a picture of how it would look. 334 00:17:33,100 --> 00:17:35,410 So here's the ethidium bromide. 335 00:17:35,410 --> 00:17:39,328 And here's a pair of stacked bases, this one and this one. 336 00:17:39,328 --> 00:17:40,870 And there he is, right in the middle. 337 00:17:40,870 --> 00:17:44,560 See that ring coming towards you is that. 338 00:17:44,560 --> 00:17:47,860 And then here's this thing that slides straight 339 00:17:47,860 --> 00:17:51,130 between the bases and might cause a little bit of a bulge. 340 00:17:51,130 --> 00:17:54,460 And we would call this a DNA intercalator. 341 00:17:54,460 --> 00:17:56,650 It slides into the DNA. 342 00:17:56,650 --> 00:17:59,170 And you can see over here, the structure of DNA. 343 00:17:59,170 --> 00:18:01,630 So you could picture ethidium bromide 344 00:18:01,630 --> 00:18:03,770 sliding between the bases. 345 00:18:03,770 --> 00:18:06,100 Now, there's a big problem here. 346 00:18:06,100 --> 00:18:10,630 Because you can't use DNA ethidium bromide. 347 00:18:10,630 --> 00:18:11,968 It's pretty toxic. 348 00:18:11,968 --> 00:18:12,885 Why would it be toxic? 349 00:18:18,210 --> 00:18:19,700 Well, no, it's not the bromide. 350 00:18:19,700 --> 00:18:22,880 It's the fact, more, think of what the dye does 351 00:18:22,880 --> 00:18:24,350 when it gets to the DNA. 352 00:18:24,350 --> 00:18:27,290 What would that do to things like replication 353 00:18:27,290 --> 00:18:28,850 and transcription? 354 00:18:28,850 --> 00:18:30,620 It just kind of messes it up. 355 00:18:30,620 --> 00:18:34,910 And so these are toxic dyes that can only be used in fixed cells 356 00:18:34,910 --> 00:18:37,500 to do observations of cells. 357 00:18:37,500 --> 00:18:39,110 So we use it a lot. 358 00:18:39,110 --> 00:18:41,600 We need to be careful of it because if it 359 00:18:41,600 --> 00:18:45,600 gets absorbed through our skin, it could get into our cells. 360 00:18:45,600 --> 00:18:47,630 And it could interfere with replication 361 00:18:47,630 --> 00:18:49,430 and other cellular processes. 362 00:18:49,430 --> 00:18:51,980 Because it would accumulate on our cellular DNA. 363 00:18:57,630 --> 00:19:00,350 And in fact, the interesting thing 364 00:19:00,350 --> 00:19:03,020 that a lot of molecules that actually 365 00:19:03,020 --> 00:19:05,630 have these sort of flat, pancake shapes 366 00:19:05,630 --> 00:19:08,000 with lots of double bonds are actually 367 00:19:08,000 --> 00:19:10,130 pretty important in biology. 368 00:19:10,130 --> 00:19:13,490 Because they end up being chemotherapeutic agents. 369 00:19:13,490 --> 00:19:15,890 So what I've shown you here is what's 370 00:19:15,890 --> 00:19:18,230 known as an anthracycline structure. 371 00:19:18,230 --> 00:19:20,870 I believe this is adriamycin, I could be off. 372 00:19:20,870 --> 00:19:26,760 But it's a natural product that's isolated from bacteria. 373 00:19:26,760 --> 00:19:29,210 And it has this structure that also 374 00:19:29,210 --> 00:19:31,880 makes it a DNA intercalator. 375 00:19:31,880 --> 00:19:35,240 And it's used as a cancer chemotherapeutic agent 376 00:19:35,240 --> 00:19:39,290 because it interferes with cell division and proliferation. 377 00:19:39,290 --> 00:19:42,140 So we actually exploit that property. 378 00:19:42,140 --> 00:19:46,670 But only with cells that we want to kill or stop dividing. 379 00:19:46,670 --> 00:19:48,320 So you could picture, well, I don't 380 00:19:48,320 --> 00:19:51,110 want to use something that's going 381 00:19:51,110 --> 00:19:53,810 to interfere with cells if I'm doing live cell imaging. 382 00:19:53,810 --> 00:19:56,120 Because I'm going to have trouble with the properties 383 00:19:56,120 --> 00:19:57,128 of the cells. 384 00:19:57,128 --> 00:19:58,670 So fluorophores are great, but you've 385 00:19:58,670 --> 00:20:03,200 got to worry about them because they can get transferred 386 00:20:03,200 --> 00:20:05,840 through the skin, through cellular membranes 387 00:20:05,840 --> 00:20:08,060 because they're often quite greasy. 388 00:20:08,060 --> 00:20:10,070 And they can get in and interfere 389 00:20:10,070 --> 00:20:12,650 with essential processes of DNA. 390 00:20:12,650 --> 00:20:16,220 So because of this, there's been quite a revolution in the work 391 00:20:16,220 --> 00:20:20,270 done with DNA binding agents that bind a little differently 392 00:20:20,270 --> 00:20:22,280 and are way less toxic. 393 00:20:22,280 --> 00:20:25,760 So I want to describe to you a series of dyes that are known 394 00:20:25,760 --> 00:20:32,350 as DAPI and HOECHST, H-O-E-C-H-S-T. This was, 395 00:20:32,350 --> 00:20:36,830 I believe, discovered in a bio company in Germany. 396 00:20:36,830 --> 00:20:40,670 And these are different kinds of dyes that 397 00:20:40,670 --> 00:20:43,130 fluoresce on binding to DNA. 398 00:20:43,130 --> 00:20:45,890 So they are still useful in that same context. 399 00:20:45,890 --> 00:20:49,100 You can in fact substitute ethidium bromide 400 00:20:49,100 --> 00:20:50,390 with these dyes. 401 00:20:50,390 --> 00:20:53,000 But they bind to DNA pretty differently. 402 00:20:53,000 --> 00:20:55,430 And I want you to take a look at these pictures. 403 00:20:55,430 --> 00:20:59,810 So here in green and blue-- and apologies to the color blind-- 404 00:20:59,810 --> 00:21:06,350 you see a molecule of this compound here bound to DNA. 405 00:21:06,350 --> 00:21:10,100 So it's pretty clear it's different from intercalation, 406 00:21:10,100 --> 00:21:11,060 right? 407 00:21:11,060 --> 00:21:16,080 You can see it's more sliding around one part of the DNA. 408 00:21:16,080 --> 00:21:19,370 And when those molecules bind to DNA in water, 409 00:21:19,370 --> 00:21:20,570 they don't fluorescence. 410 00:21:20,570 --> 00:21:24,410 When they bind to DNA they fluorescent an intense cyan 411 00:21:24,410 --> 00:21:25,700 blue. 412 00:21:25,700 --> 00:21:28,205 So that's at a shorter wavelength from the ethidium 413 00:21:28,205 --> 00:21:30,030 bromide. 414 00:21:30,030 --> 00:21:31,910 So taking a look at this structure, 415 00:21:31,910 --> 00:21:36,140 does anyone want to explain to me how the molecules might 416 00:21:36,140 --> 00:21:38,450 bind to DNA? 417 00:21:38,450 --> 00:21:41,270 We wouldn't call it intercalation. 418 00:21:41,270 --> 00:21:46,190 We know intercalation is perpendicular to the axis 419 00:21:46,190 --> 00:21:47,550 of the DNA. 420 00:21:47,550 --> 00:21:51,530 So where, looking at this, do you think these bind? 421 00:21:51,530 --> 00:21:54,710 A while ago when I was talking about the structure of DNA, 422 00:21:54,710 --> 00:22:00,050 I like to think of DNA as having two grooves, two places where 423 00:22:00,050 --> 00:22:01,280 things combine to it. 424 00:22:01,280 --> 00:22:03,740 And that's a minor groove. 425 00:22:03,740 --> 00:22:07,190 And then this big trench is what's called the major groove. 426 00:22:07,190 --> 00:22:09,740 And certain molecules bind in one groove. 427 00:22:09,740 --> 00:22:11,437 And other molecules bind in the other. 428 00:22:11,437 --> 00:22:12,770 Where do you think it's binding? 429 00:22:12,770 --> 00:22:13,850 Just by inspection. 430 00:22:16,540 --> 00:22:17,040 Yeah. 431 00:22:17,040 --> 00:22:18,110 AUDIENCE: Looks like it's in the minor groove. 432 00:22:18,110 --> 00:22:21,200 PROFESSOR: That's correct, it's just snuggled just perfectly 433 00:22:21,200 --> 00:22:22,105 in the minor groove. 434 00:22:22,105 --> 00:22:23,480 If it was in the major groove, it 435 00:22:23,480 --> 00:22:25,310 would be swimming around in that groove. 436 00:22:25,310 --> 00:22:27,290 It's almost too big. 437 00:22:27,290 --> 00:22:29,810 So what's really cool about these dyes 438 00:22:29,810 --> 00:22:34,640 is they slide in between into the minor groove. 439 00:22:34,640 --> 00:22:38,470 And they also make some contacts with the phosphodiester 440 00:22:38,470 --> 00:22:38,970 backbone. 441 00:22:38,970 --> 00:22:41,990 But it's not that they're dancing on the phosphodiester 442 00:22:41,990 --> 00:22:42,740 backbone. 443 00:22:42,740 --> 00:22:44,630 They're literally in the groove. 444 00:22:44,630 --> 00:22:48,290 But there's some opportunity for electrostatic interactions. 445 00:22:48,290 --> 00:22:51,380 And so these compounds would be known 446 00:22:51,380 --> 00:22:53,480 to bind in the minor groove. 447 00:22:53,480 --> 00:22:56,510 And in fact, they bind in particular regions of DNA 448 00:22:56,510 --> 00:22:59,030 where there's AT, not GC. 449 00:22:59,030 --> 00:23:01,020 Those are the places where there's just 450 00:23:01,020 --> 00:23:03,890 the pair of hydrogen bonds instead of the trio. 451 00:23:03,890 --> 00:23:07,310 That's just their habit, their personality. 452 00:23:07,310 --> 00:23:10,760 So chemistry was very important here. 453 00:23:10,760 --> 00:23:13,370 You had a good dye, but it was toxic. 454 00:23:13,370 --> 00:23:15,440 But improved dyes came along that could 455 00:23:15,440 --> 00:23:18,800 be used in living systems that are not toxic. 456 00:23:18,800 --> 00:23:20,690 Because if you bind in those grooves 457 00:23:20,690 --> 00:23:22,670 and you're dissociating easily, you're 458 00:23:22,670 --> 00:23:25,660 not going to interfere so much with replication. 459 00:23:25,660 --> 00:23:27,500 Does that make sense? 460 00:23:27,500 --> 00:23:29,120 So it's a weaker force. 461 00:23:29,120 --> 00:23:31,970 It's not going to have a big detrimental effect. 462 00:23:31,970 --> 00:23:33,620 Now, I moved this slide up. 463 00:23:33,620 --> 00:23:36,770 I realized he was in the wrong place in the deck. 464 00:23:36,770 --> 00:23:42,700 This is just an application of the DNA minor groove binder 465 00:23:42,700 --> 00:23:43,640 CEOCHST. 466 00:23:43,640 --> 00:23:47,450 And in this case, we're looking at three cells. 467 00:23:47,450 --> 00:23:51,350 These two are not actively dividing. 468 00:23:51,350 --> 00:23:53,540 But take a look at this cell, it's 469 00:23:53,540 --> 00:23:59,480 actually clearly in the state preparing for cell division. 470 00:23:59,480 --> 00:24:04,290 And what you can see here is that in the nucleus, 471 00:24:04,290 --> 00:24:07,610 the DNA is pretty diffuse before things really 472 00:24:07,610 --> 00:24:12,980 start to condense and line up for DNA replication and cell 473 00:24:12,980 --> 00:24:13,940 division. 474 00:24:13,940 --> 00:24:16,970 And what's intriguing to me is that this rather 475 00:24:16,970 --> 00:24:19,850 diffuse blue dye, that's probably a bit 476 00:24:19,850 --> 00:24:22,400 more loosely associated with DNA, 477 00:24:22,400 --> 00:24:25,520 becomes much clearer and brighter when 478 00:24:25,520 --> 00:24:29,070 the chromosomes are in the state they're in for cell division. 479 00:24:29,070 --> 00:24:30,940 So you can see them here. 480 00:24:30,940 --> 00:24:32,667 And one question here. 481 00:24:32,667 --> 00:24:34,250 So, if you're looking at cells, you're 482 00:24:34,250 --> 00:24:36,860 trying to observe cells, where else in the cell 483 00:24:36,860 --> 00:24:37,955 are you going to see DNA? 484 00:24:40,540 --> 00:24:42,730 So we can see the nuclear DNA, we 485 00:24:42,730 --> 00:24:45,100 can see what stage of the cell cycle it's in-- 486 00:24:45,100 --> 00:24:45,790 Yeah, Carmen. 487 00:24:45,790 --> 00:24:46,630 AUDIENCE: The mitochondria. 488 00:24:46,630 --> 00:24:48,172 PROFESSOR: Yeah, in the mitochondria. 489 00:24:48,172 --> 00:24:51,230 So you could also spot that within the cell 490 00:24:51,230 --> 00:24:53,860 if you're at a sufficient amplification. 491 00:24:53,860 --> 00:24:57,220 So you know that there's not DNA running around everywhere. 492 00:24:57,220 --> 00:25:01,330 It's literally in very specific places with the cell. 493 00:25:01,330 --> 00:25:06,010 These dyes will bind also to other nucleic acids. 494 00:25:06,010 --> 00:25:07,540 But they don't bind so well. 495 00:25:07,540 --> 00:25:10,660 Because those don't have the really repetitive, 496 00:25:10,660 --> 00:25:14,770 double stranded nucleotide structures. 497 00:25:14,770 --> 00:25:18,820 But there are other dyes that bind much more specifically 498 00:25:18,820 --> 00:25:19,600 to RNA. 499 00:25:19,600 --> 00:25:21,040 But we won't discuss them. 500 00:25:21,040 --> 00:25:22,930 So nucleic acids seem to be something 501 00:25:22,930 --> 00:25:25,970 that we can definitely pinpoint with fluorescence. 502 00:25:25,970 --> 00:25:27,310 We can see where it is. 503 00:25:27,310 --> 00:25:29,410 We could follow cell division. 504 00:25:29,410 --> 00:25:32,380 We could look to see the progress of cell division. 505 00:25:32,380 --> 00:25:36,430 For example, upon adding things to a cell, can you see-- 506 00:25:36,430 --> 00:25:39,580 remember very, very early on, we showed you 507 00:25:39,580 --> 00:25:41,410 movies of cells dividing. 508 00:25:41,410 --> 00:25:43,780 You could do that with this kind of dye 509 00:25:43,780 --> 00:25:46,960 because it's a non-toxic dye. 510 00:25:46,960 --> 00:25:49,780 So, great, so far, so good. 511 00:25:49,780 --> 00:25:52,870 So the key thing, though, about biology 512 00:25:52,870 --> 00:25:56,200 is we have so many other entities within a cell 513 00:25:56,200 --> 00:25:59,080 that we want to be able to track and monitor. 514 00:25:59,080 --> 00:26:02,500 And what we needed, what is absolutely essential 515 00:26:02,500 --> 00:26:05,720 are reagents to do that. 516 00:26:05,720 --> 00:26:18,900 So I want to talk to you about biological tools of monitoring. 517 00:26:21,978 --> 00:26:23,145 And you know what these are? 518 00:26:23,145 --> 00:26:26,520 These are antibodies, monitoring proteins. 519 00:26:26,520 --> 00:26:30,120 And in fact, you can also coax antibodies 520 00:26:30,120 --> 00:26:31,650 to recognize carbohydrates. 521 00:26:31,650 --> 00:26:33,330 So I'm going to just put these here. 522 00:26:40,500 --> 00:26:43,270 But they're a little bit harder to bind to antibodies. 523 00:26:43,270 --> 00:26:44,980 But nevertheless, those are useful. 524 00:26:44,980 --> 00:26:48,620 So we're going to talk now about antibodies, 525 00:26:48,620 --> 00:26:52,225 which are agents of the human adaptive immune system. 526 00:27:10,620 --> 00:27:16,350 And how they have been exploited intensively to study biology. 527 00:27:16,350 --> 00:27:20,880 Now, what you will learn from Professor Martin in two 528 00:27:20,880 --> 00:27:23,310 or three lectures time is much more 529 00:27:23,310 --> 00:27:26,340 about the nuts and bolts of the immune cells, 530 00:27:26,340 --> 00:27:27,870 of the immune system. 531 00:27:27,870 --> 00:27:32,010 And how it mounts a response to disease and other features. 532 00:27:32,010 --> 00:27:35,160 I'm going to focus completely on the technological side 533 00:27:35,160 --> 00:27:37,470 of antibodies and how they are useful 534 00:27:37,470 --> 00:27:39,990 reagents to study biology. 535 00:27:39,990 --> 00:27:44,460 Because if you want to recognize a protein in a cell, 536 00:27:44,460 --> 00:27:48,270 you need a particular entity that will bind to that protein 537 00:27:48,270 --> 00:27:50,850 and show you where it is through some kind of signal, 538 00:27:50,850 --> 00:27:52,680 for example, fluorescence. 539 00:27:52,680 --> 00:27:55,650 So what I want to do is give you the minimal description 540 00:27:55,650 --> 00:27:58,230 of antibodies so you can understand this. 541 00:27:58,230 --> 00:28:00,600 But later you're going to revisit it 542 00:28:00,600 --> 00:28:02,730 in a bit more complicated venue. 543 00:28:02,730 --> 00:28:04,830 But for the time being, I'm just going 544 00:28:04,830 --> 00:28:12,970 to talk about B cells, which are cells that produce antibodies. 545 00:28:17,090 --> 00:28:19,580 And I'm going to talk to you about how 546 00:28:19,580 --> 00:28:22,730 they recognize their targets. 547 00:28:22,730 --> 00:28:25,850 Because what we have in the adaptive immune system 548 00:28:25,850 --> 00:28:30,530 is an amazing system where you can do combinatorial biology 549 00:28:30,530 --> 00:28:33,420 and basically recognize any target entity 550 00:28:33,420 --> 00:28:34,730 you're interested in. 551 00:28:34,730 --> 00:28:37,790 So let's take a look at this, keeping in mind 552 00:28:37,790 --> 00:28:40,490 that this is us exploiting biology 553 00:28:40,490 --> 00:28:42,920 to make reagents to do biology. 554 00:28:42,920 --> 00:28:45,740 It's kind of a cool sort of cyclical process. 555 00:28:45,740 --> 00:28:49,940 So the cells of the hematopoietic immune-- 556 00:28:49,940 --> 00:28:52,910 sorry the hematopoietic system, those are the ones that are 557 00:28:52,910 --> 00:28:57,680 important in blood cells, form a lot of different-- 558 00:28:57,680 --> 00:28:59,750 wait a minute, I'm on triple-- 559 00:28:59,750 --> 00:29:00,960 triple tools here. 560 00:29:00,960 --> 00:29:02,660 There are a bunch of different cells 561 00:29:02,660 --> 00:29:05,840 that are produced from the pluripotent hematopoietic 562 00:29:05,840 --> 00:29:06,480 cells. 563 00:29:06,480 --> 00:29:09,410 They're either the white cells or the red cell types. 564 00:29:09,410 --> 00:29:13,470 But what we're going to focus right in on are the B cells. 565 00:29:13,470 --> 00:29:15,530 These are the cells of the immune system that 566 00:29:15,530 --> 00:29:18,350 produce soluble antibodies, OK. 567 00:29:21,260 --> 00:29:25,160 And when you challenge a B cell population 568 00:29:25,160 --> 00:29:29,330 with a foreign entity, the B cell population 569 00:29:29,330 --> 00:29:32,560 will go into gear to produce antibodies 570 00:29:32,560 --> 00:29:37,220 that very specifically recognize that foreign target, because 571 00:29:37,220 --> 00:29:39,800 in the human adaptive immune system, that 572 00:29:39,800 --> 00:29:44,190 might be a wonderful tool to get rid of that foreign entity. 573 00:29:44,190 --> 00:29:45,830 So we're going to focus exclusively 574 00:29:45,830 --> 00:29:49,430 on the B cells and the way that they mature 575 00:29:49,430 --> 00:29:52,760 to produce soluble antibodies, those are down at the bottom 576 00:29:52,760 --> 00:29:56,120 here, based on what they've been challenged with. 577 00:29:56,120 --> 00:29:58,880 So what this little schematic shows you is you 578 00:29:58,880 --> 00:30:02,780 have a bunch of different B cells. 579 00:30:02,780 --> 00:30:05,150 And there's something you want to recognize. 580 00:30:05,150 --> 00:30:08,770 Let's just say it's a molecule-- 581 00:30:08,770 --> 00:30:11,540 an EGF molecule, a cytokine. 582 00:30:11,540 --> 00:30:15,170 What you might do is challenge this population 583 00:30:15,170 --> 00:30:16,700 with the cytokine. 584 00:30:16,700 --> 00:30:20,630 Only one B cell type will bind to the cytokine. 585 00:30:20,630 --> 00:30:23,130 And then that will get amplified. 586 00:30:23,130 --> 00:30:25,700 And then you will end up with B cells 587 00:30:25,700 --> 00:30:28,010 that produce a lot of an antibody 588 00:30:28,010 --> 00:30:31,610 to a cytokine such as EGF. 589 00:30:31,610 --> 00:30:34,280 I'm truly dumbing this down. 590 00:30:34,280 --> 00:30:36,140 I just want you to get the gist of it 591 00:30:36,140 --> 00:30:38,750 for the purpose of this discussion. 592 00:30:38,750 --> 00:30:42,940 Now B cells adopt a very classical shape. 593 00:30:42,940 --> 00:30:47,270 And I'm just going to show you the quaternary structure of a B 594 00:30:47,270 --> 00:30:47,810 cell-- 595 00:30:47,810 --> 00:30:54,140 of an antibody in linear form here. 596 00:30:54,140 --> 00:30:56,630 So it doesn't look very exciting right now. 597 00:30:56,630 --> 00:31:06,040 There are two light chains, which I've just 598 00:31:06,040 --> 00:31:08,410 shown in schematic form. 599 00:31:08,410 --> 00:31:11,440 These are just polypeptide chains. 600 00:31:11,440 --> 00:31:13,060 And there are two heavy chains. 601 00:31:19,720 --> 00:31:22,820 All right, so that's their basic structure. 602 00:31:22,820 --> 00:31:25,940 It's held together in a stable quarternary 603 00:31:25,940 --> 00:31:28,940 structure with this complex. 604 00:31:28,940 --> 00:31:33,200 And there may be disulfides across and throughout 605 00:31:33,200 --> 00:31:34,280 the structure. 606 00:31:34,280 --> 00:31:36,530 Now what's so special about antibodies? 607 00:31:36,530 --> 00:31:38,360 They're pretty big molecules. 608 00:31:38,360 --> 00:31:40,100 The molecular weight is pretty high. 609 00:31:44,520 --> 00:31:46,700 But the key thing about antibodies 610 00:31:46,700 --> 00:31:53,910 is that the majority of the structure 611 00:31:53,910 --> 00:31:55,455 stays fairly constant. 612 00:31:58,080 --> 00:31:59,300 So I'm just going to-- 613 00:31:59,300 --> 00:32:05,760 so this doesn't get modified when B cells mature. 614 00:32:05,760 --> 00:32:10,640 But another part of the structure is variable. 615 00:32:14,680 --> 00:32:17,680 And when B cells mature, there's loads 616 00:32:17,680 --> 00:32:20,860 of rearranging in that variable section in order 617 00:32:20,860 --> 00:32:24,050 that it adapt to bind to target. 618 00:32:24,050 --> 00:32:26,890 So what you've seen here is that the target-- 619 00:32:26,890 --> 00:32:28,660 see if this little fellow works anymore. 620 00:32:28,660 --> 00:32:29,160 No. 621 00:32:29,160 --> 00:32:30,730 Ah. 622 00:32:30,730 --> 00:32:32,890 What you see here is the light chain 623 00:32:32,890 --> 00:32:35,990 in green, heavy chain in blue. 624 00:32:35,990 --> 00:32:37,460 And it's a double version of it. 625 00:32:37,460 --> 00:32:39,910 We always draw antibodies as this V shape. 626 00:32:39,910 --> 00:32:49,890 And an antigen-- you've heard this word before-- 627 00:32:49,890 --> 00:32:53,625 it's a foreign entity that's foreign to the immune system. 628 00:33:02,490 --> 00:33:08,390 The antigen binding site is right here 629 00:33:08,390 --> 00:33:11,890 at the tips of the antibody. 630 00:33:11,890 --> 00:33:15,200 And I think the picture up there is kind of clearer. 631 00:33:15,200 --> 00:33:16,540 Let's put that forward again. 632 00:33:16,540 --> 00:33:19,630 And you can see very specifically the structure. 633 00:33:19,630 --> 00:33:22,120 The C's designate constant regions. 634 00:33:27,930 --> 00:33:30,360 See C all the way through here. 635 00:33:30,360 --> 00:33:34,290 And V's represent variable regions, which I've shown you. 636 00:33:34,290 --> 00:33:37,620 And at the tip of the V's are the antigen binding sites. 637 00:33:37,620 --> 00:33:39,780 So you're going to see more about antibodies 638 00:33:39,780 --> 00:33:41,252 in the immune system. 639 00:33:41,252 --> 00:33:42,960 But what you want to accept is that these 640 00:33:42,960 --> 00:33:47,040 are biological macromolecules that particularly evolved 641 00:33:47,040 --> 00:33:49,710 to recognize target antigens. 642 00:33:49,710 --> 00:33:55,660 And you can use them reliably as biological reagents. 643 00:33:55,660 --> 00:33:59,110 All right, so how do you achieve the diversity? 644 00:33:59,110 --> 00:34:02,290 There are hundreds of thousands of different antibodies 645 00:34:02,290 --> 00:34:03,850 in the human system. 646 00:34:03,850 --> 00:34:07,540 If we had a gene for every single different light 647 00:34:07,540 --> 00:34:11,080 chain and every heavy chain, you know, our DNA 648 00:34:11,080 --> 00:34:13,090 would be completely swamped by being 649 00:34:13,090 --> 00:34:17,679 dedicated to the genetic material for antibodies. 650 00:34:17,679 --> 00:34:20,949 So instead there is a particular system 651 00:34:20,949 --> 00:34:25,840 which provides little portions of the DNA structure 652 00:34:25,840 --> 00:34:29,210 that are in little pieces of variable components 653 00:34:29,210 --> 00:34:33,440 that can get zipped together through transcription 654 00:34:33,440 --> 00:34:37,810 and slicing events to give you a bunch of antibodies that have 655 00:34:37,810 --> 00:34:40,389 different variable regions. 656 00:34:40,389 --> 00:34:42,850 And this is what's known as the BDJ system. 657 00:34:42,850 --> 00:34:46,120 And you'll hear more about that from Professor Martin. 658 00:34:46,120 --> 00:34:48,070 So basically what you want to think about 659 00:34:48,070 --> 00:34:52,659 is it's a combinatorial system to take little pieces of DNA 660 00:34:52,659 --> 00:34:55,510 into a super molecular structure to give you 661 00:34:55,510 --> 00:34:59,320 antibody combining sites that can recognize virtually 662 00:34:59,320 --> 00:35:00,880 any target. 663 00:35:00,880 --> 00:35:02,480 Anyone got any questions here? 664 00:35:02,480 --> 00:35:06,630 I'm seeing a few worried faces. 665 00:35:06,630 --> 00:35:08,550 Somebody ask me a question if you 666 00:35:08,550 --> 00:35:10,830 feel I could clarify a component of this 667 00:35:10,830 --> 00:35:11,970 or are you OK with this? 668 00:35:15,610 --> 00:35:18,150 Anybody? 669 00:35:18,150 --> 00:35:20,060 OK, I'll move forward. 670 00:35:23,050 --> 00:35:25,600 So we talked about antibodies. 671 00:35:25,600 --> 00:35:27,670 When you get a population of B cells 672 00:35:27,670 --> 00:35:33,550 that produce antibodies to a particular target, 673 00:35:33,550 --> 00:35:36,070 these may be what are known as polyclonal-- 674 00:35:41,283 --> 00:35:43,700 a polyclonal-- sorry, I should have written that up here-- 675 00:35:43,700 --> 00:35:52,970 a polyclonal antibody, as might be suggested by the name, 676 00:35:52,970 --> 00:35:54,350 is an antibody-- 677 00:35:54,350 --> 00:35:58,460 let's say it recognizes a molecular entity. 678 00:35:58,460 --> 00:36:00,010 So the antibodies-- 679 00:36:00,010 --> 00:36:03,520 I'm going to draw them as little y shaped molecules-- 680 00:36:03,520 --> 00:36:09,970 may recognize different parts of the antigen. So antibody A, 681 00:36:09,970 --> 00:36:13,660 B, C recognize different parts of the antigen. 682 00:36:13,660 --> 00:36:15,280 Those would be polyclonal. 683 00:36:15,280 --> 00:36:19,750 It would be a mixed bag of antibody molecules 684 00:36:19,750 --> 00:36:22,750 that shows specificity for a target. 685 00:36:22,750 --> 00:36:27,156 But people also tend to use a great deal 686 00:36:27,156 --> 00:36:34,700 of monoclonal antibodies, because they 687 00:36:34,700 --> 00:36:36,500 are a lot more specific. 688 00:36:36,500 --> 00:36:40,780 So if you had a selection of polyclonal antibodies, 689 00:36:40,780 --> 00:36:44,330 a monoclonal antibody would be a single population 690 00:36:44,330 --> 00:36:47,600 that recognizes a single antigen or epitope 691 00:36:47,600 --> 00:36:49,520 in your antigenic molecule. 692 00:36:49,520 --> 00:36:53,510 And the way those are made is through the engineering method 693 00:36:53,510 --> 00:36:57,170 where you fuse spleen cells with myeloma cells. 694 00:36:57,170 --> 00:36:59,360 And then you get a hybrid-- 695 00:36:59,360 --> 00:37:03,050 what are known as hybridomas that produce very specifically 696 00:37:03,050 --> 00:37:05,210 just monoclonal antibodies. 697 00:37:05,210 --> 00:37:07,850 So that's a bit of background there about the antibodies. 698 00:37:07,850 --> 00:37:11,240 You'll see it on the rerun when Professor Martin talks. 699 00:37:11,240 --> 00:37:14,150 But I just wanted to give you a bit of exposure to this. 700 00:37:14,150 --> 00:37:18,360 So let's now look at how antibodies can be useful. 701 00:37:18,360 --> 00:37:21,290 So let's say you want to visualize in a cell-- let's 702 00:37:21,290 --> 00:37:24,050 move straight to a real targeted application. 703 00:37:24,050 --> 00:37:27,680 We want to make an antibody that might recognize actin 704 00:37:27,680 --> 00:37:31,100 and a different antibody that might recognize tubulin 705 00:37:31,100 --> 00:37:35,930 to take a look at cells through the use of antibody structures. 706 00:37:35,930 --> 00:37:39,860 The way you generate antibodies is through laboratory animals. 707 00:37:39,860 --> 00:37:43,700 And very commonly we use either mice or rabbits 708 00:37:43,700 --> 00:37:45,290 for antibody production. 709 00:37:47,810 --> 00:37:51,830 The rabbit is used when you need a lot more antibody material. 710 00:37:51,830 --> 00:37:55,020 The mouse will satisfy for some experiments. 711 00:37:55,020 --> 00:37:59,970 So the way you make antibodies is by injecting. 712 00:37:59,970 --> 00:38:03,530 So this would be the foreign agent or antigen 713 00:38:03,530 --> 00:38:05,570 that you want to make an antibody to, 714 00:38:05,570 --> 00:38:09,020 which would normally bind at the variable region 715 00:38:09,020 --> 00:38:11,390 of this immunoglobulin molecule. 716 00:38:11,390 --> 00:38:14,510 You inject the mouse with that antigen. Excuse 717 00:38:14,510 --> 00:38:15,533 me-- that's a rabbit. 718 00:38:15,533 --> 00:38:17,450 You know, I haven't been in biology very long. 719 00:38:17,450 --> 00:38:20,430 But I can tell that's a rabbit, right? 720 00:38:20,430 --> 00:38:26,185 Anyway, so you inject the rabbit with a human protein. 721 00:38:26,185 --> 00:38:27,560 If you-- what would happen if you 722 00:38:27,560 --> 00:38:35,450 injected the rabbit with rabbit actin or with rabbit tubulin? 723 00:38:35,450 --> 00:38:40,600 What would you-- would you expect to see a response? 724 00:38:40,600 --> 00:38:41,430 No, right? 725 00:38:41,430 --> 00:38:42,150 Yeah. 726 00:38:42,150 --> 00:38:45,840 Because the organisms are adapted 727 00:38:45,840 --> 00:38:48,510 not to recognize their own proteins 728 00:38:48,510 --> 00:38:51,850 unless there's some disorder like an autoimmune disease. 729 00:38:51,850 --> 00:38:56,470 So you would inject the rabbit with a human epitope, 730 00:38:56,470 --> 00:39:00,300 so for example human actin, generate antibodies 731 00:39:00,300 --> 00:39:03,150 with a specificity for actin, and then 732 00:39:03,150 --> 00:39:06,930 you would label those antibodies with a fluorescent marker, 733 00:39:06,930 --> 00:39:10,290 so you could track it or follow it through chemistry. 734 00:39:10,290 --> 00:39:13,620 Alternatively, you might want to make a different antibody 735 00:39:13,620 --> 00:39:14,970 for tubulin. 736 00:39:14,970 --> 00:39:18,750 And then you would differentiate the antibody against tubulin 737 00:39:18,750 --> 00:39:21,720 from the antibody against actin by labeling it 738 00:39:21,720 --> 00:39:24,000 with a different color fluorophore. 739 00:39:24,000 --> 00:39:27,390 So you've really got two types of macromolecule 740 00:39:27,390 --> 00:39:30,210 that can be interacted with a fixed cell 741 00:39:30,210 --> 00:39:32,490 and recognize those two macromolecules 742 00:39:32,490 --> 00:39:33,570 within the protein. 743 00:39:33,570 --> 00:39:35,670 So what's important here is that you 744 00:39:35,670 --> 00:39:38,400 don't use the protein from-- 745 00:39:38,400 --> 00:39:40,590 if you want to study human cells, 746 00:39:40,590 --> 00:39:43,800 you use antibodies produced in rabbit or mouse. 747 00:39:43,800 --> 00:39:45,900 If you want to study rabbit cells, 748 00:39:45,900 --> 00:39:48,500 you could produce it in different organisms. 749 00:39:48,500 --> 00:39:52,250 You're not going to produce the antibodies in the same host. 750 00:39:52,250 --> 00:39:53,900 Nowadays-- yes. 751 00:39:53,900 --> 00:39:56,854 AUDIENCE: How do you know that you have the correct antibody, 752 00:39:56,854 --> 00:40:00,600 like if that is the right one? 753 00:40:00,600 --> 00:40:01,770 PROFESSOR: Right. 754 00:40:01,770 --> 00:40:05,220 OK, so you would prepare the-- 755 00:40:05,220 --> 00:40:07,050 you would do them. 756 00:40:07,050 --> 00:40:09,150 There's a lot of screening takes place. 757 00:40:09,150 --> 00:40:11,670 So you inject the mouse or rabbit. 758 00:40:11,670 --> 00:40:13,260 And then you collect the serum. 759 00:40:13,260 --> 00:40:15,510 And you look for whether the serum 760 00:40:15,510 --> 00:40:17,820 gets enriched and enriched in antibodies 761 00:40:17,820 --> 00:40:20,040 that recognize the target. 762 00:40:20,040 --> 00:40:23,280 So you'll see an increase in what's known as the titer. 763 00:40:23,280 --> 00:40:26,040 And then once you get a high titer, you'll-- 764 00:40:26,040 --> 00:40:28,950 unfortunately, you can just collect the serum 765 00:40:28,950 --> 00:40:31,440 or depending if you want to make monoclonal, 766 00:40:31,440 --> 00:40:34,380 you'd have to sacrifice the animal to get the spleen cells. 767 00:40:34,380 --> 00:40:36,660 But then you collect the antibodies 768 00:40:36,660 --> 00:40:38,340 through an affinity method that's 769 00:40:38,340 --> 00:40:40,380 directed just at antibodies. 770 00:40:40,380 --> 00:40:41,790 Then you've got your population. 771 00:40:41,790 --> 00:40:44,400 And you can throw a fluorophore dye at it 772 00:40:44,400 --> 00:40:45,600 and chemically label it. 773 00:40:45,600 --> 00:40:47,280 So it's a good point there. 774 00:40:47,280 --> 00:40:48,840 There's a lot of work being done now 775 00:40:48,840 --> 00:40:51,660 with antibodies from different organisms, in fact, 776 00:40:51,660 --> 00:40:53,700 you'll see them from camels. 777 00:40:53,700 --> 00:40:55,743 And they're also ones from shark. 778 00:40:55,743 --> 00:40:57,660 And the reason why they're kind of interesting 779 00:40:57,660 --> 00:41:00,630 is that they sort of have mini antibodies that are much more 780 00:41:00,630 --> 00:41:02,590 useful for technology. 781 00:41:02,590 --> 00:41:05,020 So let's see what we can do here. 782 00:41:05,020 --> 00:41:08,100 We can do fluorescence experiments. 783 00:41:08,100 --> 00:41:10,450 In order to do this with an antibody. 784 00:41:10,450 --> 00:41:13,890 And this is going to highlight a shortcoming of antibodies. 785 00:41:13,890 --> 00:41:15,420 You may take a cell that you want 786 00:41:15,420 --> 00:41:18,310 to observe different proteins in that cell, 787 00:41:18,310 --> 00:41:21,750 you have to fix the cell to make it permeable. 788 00:41:21,750 --> 00:41:25,200 Why do I need to make the cell permeable 789 00:41:25,200 --> 00:41:27,450 in order to use these antibody reagents? 790 00:41:33,330 --> 00:41:36,770 Do you think the antibodies are just going to cross-- 791 00:41:36,770 --> 00:41:38,820 DAPI gets into a cell easily. 792 00:41:38,820 --> 00:41:41,860 But what about antibodies? 793 00:41:41,860 --> 00:41:43,570 Can they cross the plasma membrane 794 00:41:43,570 --> 00:41:45,340 to get into the cell to label a target? 795 00:41:48,530 --> 00:41:49,970 What do you think? 796 00:41:49,970 --> 00:41:53,020 Who says yes? 797 00:41:53,020 --> 00:41:54,140 Who says no? 798 00:41:54,140 --> 00:41:54,750 Good. 799 00:41:54,750 --> 00:41:55,580 Thank goodness. 800 00:41:55,580 --> 00:41:56,110 OK. 801 00:41:56,110 --> 00:41:57,110 You guys don't say much. 802 00:41:57,110 --> 00:41:58,610 But I know you know the answer here. 803 00:41:58,610 --> 00:42:00,260 They just can't float into cells. 804 00:42:00,260 --> 00:42:02,430 They're too large to get into cells. 805 00:42:02,430 --> 00:42:04,940 So you have to fix the cells on a glass side 806 00:42:04,940 --> 00:42:09,940 and permeabilize them, for example, with methanol 807 00:42:09,940 --> 00:42:12,230 so that the antibodies can gain access 808 00:42:12,230 --> 00:42:14,010 to all parts of the cell. 809 00:42:14,010 --> 00:42:17,570 So here is a bright field view of cells. 810 00:42:17,570 --> 00:42:20,270 That's a little bit more specific. 811 00:42:20,270 --> 00:42:21,740 That's OK too. 812 00:42:21,740 --> 00:42:23,780 But this is what I really want to show you. 813 00:42:23,780 --> 00:42:28,280 This is what you could achieve with DAPI and an antibody 814 00:42:28,280 --> 00:42:31,190 to the actin and an antibody to tubulin. 815 00:42:31,190 --> 00:42:33,290 And you can look at the various colors 816 00:42:33,290 --> 00:42:35,000 of the fluorescence emission. 817 00:42:35,000 --> 00:42:39,650 And you see here what you've got is an anti-actin antibody 818 00:42:39,650 --> 00:42:40,580 with a red dye. 819 00:42:40,580 --> 00:42:43,850 And you can see that at the perimeters of the cells. 820 00:42:43,850 --> 00:42:47,420 You've got an anti-tubulin antibody with a green dye. 821 00:42:47,420 --> 00:42:51,980 And you can see the filamentous structure there. 822 00:42:51,980 --> 00:42:55,040 And then you've got DAPI staining bright blue 823 00:42:55,040 --> 00:42:56,290 where the nuclei are. 824 00:42:56,290 --> 00:42:58,910 So you have three unique labels that 825 00:42:58,910 --> 00:43:00,590 fluoresce at different wavelengths 826 00:43:00,590 --> 00:43:02,810 and you can directly pinpoint things. 827 00:43:02,810 --> 00:43:06,430 So fluorescence is extremely valuable for looking 828 00:43:06,430 --> 00:43:09,760 a biological systems, because we don't have a lot that 829 00:43:09,760 --> 00:43:11,120 fluoresces in the body. 830 00:43:11,120 --> 00:43:14,360 So a cell in general, if you irradiate it with light, 831 00:43:14,360 --> 00:43:17,240 you won't see any major fluorescence at all. 832 00:43:17,240 --> 00:43:21,050 So these reagents that you use to study biology, 833 00:43:21,050 --> 00:43:24,470 if they fluoresce, you've got unique signals where you can 834 00:43:24,470 --> 00:43:28,040 look at really complicated cells that may have thousands 835 00:43:28,040 --> 00:43:31,010 and thousands of proteins, sugars, nucleic acids, 836 00:43:31,010 --> 00:43:34,760 and very specifically see things by fluorescence 837 00:43:34,760 --> 00:43:38,510 because the fluorescence is a unique signal in biology 838 00:43:38,510 --> 00:43:41,960 relative to the intrinsic fluorescence of proteins 839 00:43:41,960 --> 00:43:43,730 or rather macromolecules. 840 00:43:43,730 --> 00:43:47,390 We have tryptophan and tyrosine, a couple of amino acids. 841 00:43:47,390 --> 00:43:49,670 They fluoresce, but it's so dim. 842 00:43:49,670 --> 00:43:50,870 It's nothing like this. 843 00:43:50,870 --> 00:43:53,780 You wouldn't see those kinds of signals at all. 844 00:43:53,780 --> 00:43:57,410 It's really what we call extrinsic fluorophores, 845 00:43:57,410 --> 00:44:01,190 fluorophores from outside that shine very, very brightly. 846 00:44:01,190 --> 00:44:03,770 Many of these fluorophores shine so brightly they 847 00:44:03,770 --> 00:44:07,280 can be used to look at in single molecules-- 848 00:44:07,280 --> 00:44:08,930 professor Martin described to you 849 00:44:08,930 --> 00:44:11,750 single molecule DNA sequencing. 850 00:44:11,750 --> 00:44:15,080 That actually exploits very bright fluorophores 851 00:44:15,080 --> 00:44:17,450 that is so bright that you can see just a few of them 852 00:44:17,450 --> 00:44:20,900 in one place very, very clearly. 853 00:44:20,900 --> 00:44:22,700 OK, how am I doing? 854 00:44:22,700 --> 00:44:24,800 I just want to actually leave you 855 00:44:24,800 --> 00:44:27,410 with something that's another technology. 856 00:44:27,410 --> 00:44:29,420 So you could almost picture-- 857 00:44:29,420 --> 00:44:33,460 with all of what we've seen so far, 858 00:44:33,460 --> 00:44:39,560 you could almost target any cell with an antibody that's 859 00:44:39,560 --> 00:44:42,170 specifically raised to a particular protein that's 860 00:44:42,170 --> 00:44:43,400 within the cell. 861 00:44:43,400 --> 00:44:45,260 So we can see-- 862 00:44:45,260 --> 00:44:48,210 in the next class we'll discuss what the limitations of that 863 00:44:48,210 --> 00:44:48,710 are. 864 00:44:48,710 --> 00:44:50,780 But we've already talked about the fact 865 00:44:50,780 --> 00:44:54,940 that we have to use antibodies with fixed, not living anymore, 866 00:44:54,940 --> 00:44:55,760 cells. 867 00:44:55,760 --> 00:44:59,040 So they are really dyes that can only be used in that way. 868 00:44:59,040 --> 00:45:01,220 The other place you can use fluorophores 869 00:45:01,220 --> 00:45:04,910 is for labeling DNA and from looking at DNA. 870 00:45:04,910 --> 00:45:07,880 And a particularly important technology 871 00:45:07,880 --> 00:45:12,410 is known as a DNA microarray has anybody heard of these? 872 00:45:12,410 --> 00:45:12,920 Yeah. 873 00:45:12,920 --> 00:45:17,690 So DNA microarrays absolutely exploits the complementarity 874 00:45:17,690 --> 00:45:19,080 of DNA sequences. 875 00:45:19,080 --> 00:45:22,730 So if you want to probe for a particular sequence of DNA, 876 00:45:22,730 --> 00:45:25,490 you would make the complementary strand 877 00:45:25,490 --> 00:45:27,350 and label it with a fluorophore. 878 00:45:27,350 --> 00:45:31,730 And new could, out of thousands of DNA stretches, literally 879 00:45:31,730 --> 00:45:33,830 light up the stretch of DNA that's 880 00:45:33,830 --> 00:45:36,080 complementary to your target. 881 00:45:36,080 --> 00:45:38,270 And so DNA microarrays-- 882 00:45:38,270 --> 00:45:40,040 what you see here are just the size 883 00:45:40,040 --> 00:45:42,350 of just a microscope slide. 884 00:45:42,350 --> 00:45:47,690 On this slide through arranged technologies, 885 00:45:47,690 --> 00:45:53,570 you can literally spot 40,000 distinct sequences of DNA 886 00:45:53,570 --> 00:45:55,580 in grids to recognize. 887 00:45:55,580 --> 00:45:58,400 So these DNA microarrays can be used 888 00:45:58,400 --> 00:46:04,040 for profiling genetic material or for profiling not just DNA, 889 00:46:04,040 --> 00:46:06,590 but RNA, and we'll see how at the beginning 890 00:46:06,590 --> 00:46:10,700 of the next class, in order to probe for particular stretches 891 00:46:10,700 --> 00:46:14,240 of DNA that might be disease related 892 00:46:14,240 --> 00:46:17,810 and have single nucleotide polymorphisms. 893 00:46:17,810 --> 00:46:20,870 So in order to actually just give you a little bit of a warm 894 00:46:20,870 --> 00:46:23,180 up to that, I want you to go-- 895 00:46:23,180 --> 00:46:25,640 I'm going to put a link to this in the web site. 896 00:46:25,640 --> 00:46:30,380 And it actually just shows you a virtual running of a DNA 897 00:46:30,380 --> 00:46:32,300 microarray experiments and how you 898 00:46:32,300 --> 00:46:35,090 can use it to profile disease states 899 00:46:35,090 --> 00:46:37,760 and cells versus healthy cells. 900 00:46:37,760 --> 00:46:39,930 And then at the beginning of the next class, 901 00:46:39,930 --> 00:46:42,680 I'll describe how you get information out 902 00:46:42,680 --> 00:46:44,270 of DNA microarrays. 903 00:46:44,270 --> 00:46:46,520 But at the end of the day, you're 904 00:46:46,520 --> 00:46:49,700 always using the fluorophores as the probes 905 00:46:49,700 --> 00:46:52,090 for where certain things are, OK. 906 00:46:52,090 --> 00:46:53,860 And the thing to remember here. 907 00:46:53,860 --> 00:46:56,290 Fluorescence is a magnificent tool. 908 00:46:56,290 --> 00:46:58,610 We can use fluorophores on their own. 909 00:46:58,610 --> 00:47:01,390 We can use fluorophores as attached to antibodies. 910 00:47:01,390 --> 00:47:03,400 And the DNA microarray experiments 911 00:47:03,400 --> 00:47:05,590 show you how you can use fluorophores 912 00:47:05,590 --> 00:47:08,340 attached to DNA sequences, OK. 913 00:47:08,340 --> 00:47:10,880 And I'll see you in the next class.