1 00:00:06,810 --> 00:00:09,020 PROFESSOR: Hello, and welcome to another help session on 2 00:00:09,020 --> 00:00:10,270 recombinant DNA. 3 00:00:10,270 --> 00:00:11,960 Today we're going to discuss genomic 4 00:00:11,960 --> 00:00:14,290 libraries and cDNA libraries. 5 00:00:14,290 --> 00:00:16,100 So what is a genomic library? 6 00:00:16,100 --> 00:00:19,340 Well, with most organisms the genome is really too 7 00:00:19,340 --> 00:00:20,080 large to deal with. 8 00:00:20,080 --> 00:00:22,260 It's too large to work with or to examine. 9 00:00:22,260 --> 00:00:24,870 So oftentimes it's more useful to cut it down into smaller 10 00:00:24,870 --> 00:00:27,600 pieces that you can then examine. 11 00:00:27,600 --> 00:00:29,170 How do we make a genomic library? 12 00:00:29,170 --> 00:00:32,920 Well, we start off with our cell of interest. 13 00:00:32,920 --> 00:00:34,750 And the first thing that we're going to do is we're going to 14 00:00:34,750 --> 00:00:37,940 isolate all the DNA from the cell. 15 00:00:37,940 --> 00:00:40,510 Once we have the DNA, we're going to use restriction 16 00:00:40,510 --> 00:00:43,500 enzymes to cut it into smaller sections. 17 00:00:43,500 --> 00:00:45,650 Now, we have to be very careful when we choose which 18 00:00:45,650 --> 00:00:47,720 restriction enzyme we want to use. 19 00:00:47,720 --> 00:00:51,240 We want to use an enzyme that has enough restriction sites 20 00:00:51,240 --> 00:00:53,940 in the genome so that we'll get small enough pieces to put 21 00:00:53,940 --> 00:00:56,580 in the vector, but not so small that all the 22 00:00:56,580 --> 00:00:59,960 genes get cut up. 23 00:00:59,960 --> 00:01:02,250 Usually what we we'll use for the vector is a plasmid. 24 00:01:02,250 --> 00:01:05,530 A plasmid is a very standard and easy way to get foreign 25 00:01:05,530 --> 00:01:08,030 DNA into a bacteria. 26 00:01:08,030 --> 00:01:11,440 So we're going to cut up our DNA and the plasmid with our 27 00:01:11,440 --> 00:01:12,900 restriction enzyme. 28 00:01:12,900 --> 00:01:15,390 Sometimes we might want to even make two different 29 00:01:15,390 --> 00:01:18,830 libraries using two different restriction enzymes so that 30 00:01:18,830 --> 00:01:20,125 we'll have the cuts in different places. 31 00:01:22,720 --> 00:01:26,510 Once we have our cut up DNA and our plasmids, now we need 32 00:01:26,510 --> 00:01:28,190 to ligate them together using DNA ligase. 33 00:01:30,790 --> 00:01:35,180 Once the cDNA is pasted into the plasmid, now we're ready 34 00:01:35,180 --> 00:01:38,790 to actually transform them into bacteria. 35 00:01:38,790 --> 00:01:41,620 It's very easy to transform plasmids into bacteria. 36 00:01:41,620 --> 00:01:42,990 There are multiple different ways to do it. 37 00:01:42,990 --> 00:01:46,110 One common one is to use heat shock, whereby you heat the 38 00:01:46,110 --> 00:01:49,150 bacteria up and then cool it down rapidly, which causes 39 00:01:49,150 --> 00:01:50,910 little holes to open up that allows the 40 00:01:50,910 --> 00:01:54,190 plasmids to be taken up. 41 00:01:54,190 --> 00:01:57,380 What we end up with are thousands of different 42 00:01:57,380 --> 00:02:01,280 bacteria cells, all with different sections of the DNA. 43 00:02:01,280 --> 00:02:05,170 So combined, all these different sections form the 44 00:02:05,170 --> 00:02:07,440 genome, the original genome that we have, but they're in 45 00:02:07,440 --> 00:02:08,600 much smaller sections. 46 00:02:08,600 --> 00:02:10,430 They're much easier to work with. 47 00:02:10,430 --> 00:02:14,350 And this combination of all these different cells forms 48 00:02:14,350 --> 00:02:16,590 our genomic library. 49 00:02:16,590 --> 00:02:18,470 So that's one type of library. 50 00:02:18,470 --> 00:02:21,320 Another type of library is a cDNA library. 51 00:02:21,320 --> 00:02:23,690 So the way that the cDNA library is different from a 52 00:02:23,690 --> 00:02:26,690 genomic library is that while the genomic library 53 00:02:26,690 --> 00:02:31,090 encompasses the entire genome of the cell, the cDNA library 54 00:02:31,090 --> 00:02:34,860 just looks at what genes are being expressed in the cell. 55 00:02:34,860 --> 00:02:39,870 So let's go over how we would make a cDNA library. 56 00:02:39,870 --> 00:02:42,870 What we're going to start off with is we're going to start 57 00:02:42,870 --> 00:02:45,160 off by isolating the mRNA. 58 00:02:45,160 --> 00:02:48,760 So the mRNA, clearly, is the RNA that's being 59 00:02:48,760 --> 00:02:49,690 expressed in the cell. 60 00:02:49,690 --> 00:02:51,690 This is what's ultimately-- the proteins are going to be 61 00:02:51,690 --> 00:02:54,021 produced by the cell. 62 00:02:54,021 --> 00:02:57,530 We're then going to use reverse transcription to 63 00:02:57,530 --> 00:02:59,670 create the DNA version of the RNA. 64 00:02:59,670 --> 00:03:02,750 This is what we're referring to, again, as the cDNA. 65 00:03:02,750 --> 00:03:07,700 Once we have the cDNA, we then need to add restriction sites 66 00:03:07,700 --> 00:03:08,643 to the ends of them. 67 00:03:08,643 --> 00:03:12,490 Remember, before we were just able to use innate restriction 68 00:03:12,490 --> 00:03:13,530 sites in the genomes. 69 00:03:13,530 --> 00:03:15,720 But for the cDNA library, we really want to 70 00:03:15,720 --> 00:03:17,620 keep the cDNA intact. 71 00:03:17,620 --> 00:03:19,050 We don't want to cut it up. 72 00:03:19,050 --> 00:03:22,630 So we just want to add restriction sites to the ends 73 00:03:22,630 --> 00:03:24,440 of the cDNA. 74 00:03:24,440 --> 00:03:28,190 Then we can treat the cDNA and the plasmids 75 00:03:28,190 --> 00:03:30,550 with restriction enzymes. 76 00:03:30,550 --> 00:03:34,500 Once again, once they've been cut up we can use DNA ligase 77 00:03:34,500 --> 00:03:39,850 to combine the cut up plasmids and the cut up cDNA to the 78 00:03:39,850 --> 00:03:42,930 complete plasmid plus cDNA. 79 00:03:42,930 --> 00:03:47,320 And finally, once again, we can transform our bacteria, 80 00:03:47,320 --> 00:03:50,410 and then the combination of all these cells, all with 81 00:03:50,410 --> 00:03:54,300 different types of cDNA, form our cDNA library. 82 00:03:54,300 --> 00:03:57,240 And cDNA libraries are very useful if you want to compare 83 00:03:57,240 --> 00:03:59,780 the difference in protein expressions between two 84 00:03:59,780 --> 00:04:02,010 different cells. 85 00:04:02,010 --> 00:04:02,780 Thank you very much. 86 00:04:02,780 --> 00:04:05,880 This has been another help session for recombinant DNA.