1 00:00:00,000 --> 00:00:02,000 (English captions by Andrea Matsumoto, University of Michigan.) 2 00:00:02,000 --> 00:00:10,000 The polymerase chain reaction or PCR can target and amplify any specific nucleic acid from 3 00:00:10,000 --> 00:00:13,000 complex biological samples. 4 00:00:13,000 --> 00:00:18,000 The procedure can be used for diagnosis to determine whether a clinical sample contains 5 00:00:18,000 --> 00:00:23,000 a nuclear sequence that is known to occur only in a specific pathogen. 6 00:00:23,000 --> 00:00:31,000 Or the laboratory scientists may use PCR to amplify and color large quantities of a specific 7 00:00:31,000 --> 00:00:34,000 gene for research. 8 00:00:34,000 --> 00:00:39,000 To preform PCR you must already know the sequence of the nucleic acid you wish to amplify. 9 00:00:39,000 --> 00:00:45,000 Then you define the boundaries of the target sequence by identifying short sequences at 10 00:00:45,000 --> 00:00:48,000 each end on opposite strands. 11 00:00:48,000 --> 00:00:54,000 Here, the boundaries of the target sequence are indicated by violet and green highlighting. 12 00:00:54,000 --> 00:01:00,000 If you move from these sequence in the five prime to three prime direction, the direction 13 00:01:00,000 --> 00:01:06,000 of normal DNA synthesis, the violet highlighting extends along one strand and the green highlighting 14 00:01:06,000 --> 00:01:09,000 extends along the complementary strand. 15 00:01:09,000 --> 00:01:15,000 It is difficult to show how PCR works using this double helix representation of DNA so 16 00:01:15,000 --> 00:01:22,000 the diagram with be converted to more easily understood ladder image of the DNA. 17 00:01:22,000 --> 00:01:27,000 In addition to the clinical sample, the PCR reaction requires three ingredients. 18 00:01:27,000 --> 00:01:33,000 First, there must be a massive supply of each of the four nucleotides. 19 00:01:33,000 --> 00:01:40,000 Second, the user must add a large supply of small synthetic primers that are designed 20 00:01:40,000 --> 00:01:47,000 to hybridize to the bonding sequence of either end of the targeted DNA. 21 00:01:47,000 --> 00:01:53,000 The primers are the ingredients that make the reaction specific since only DNA that 22 00:01:53,000 --> 00:01:58,000 lies between these two primers will be synthesized in the PCR reaction. 23 00:01:58,000 --> 00:02:04,000 Third, the reaction requires a DNA polymerase enzyme. 24 00:02:04,000 --> 00:02:10,000 For PCR the polymerase is actually from a bacteria that normally grows in the sea around 25 00:02:10,000 --> 00:02:14,000 hot geothermal vents on the ocean floor. 26 00:02:14,000 --> 00:02:20,000 The bacterium is called Thermus Aquaticus and the polymerase is called Taq polymerase 27 00:02:20,000 --> 00:02:21,000 for short. 28 00:02:21,000 --> 00:02:29,000 This exotic enzyme is used because it is not inactivated by the high temperatures generated 29 00:02:29,000 --> 00:02:31,000 in the PCR reaction. 30 00:02:31,000 --> 00:02:38,000 All these elements are mixed together in appropriate proportions and placed in an instrument called 31 00:02:38,000 --> 00:02:40,000 a thermocycler. 32 00:02:40,000 --> 00:02:47,000 This instrument can be programed to change the temperature of the mixture through a series 33 00:02:47,000 --> 00:02:49,000 of repetitive cycles. 34 00:02:49,000 --> 00:02:57,000 The temperature of the reaction in this demonstration is presented in the lower right panel. 35 00:02:57,000 --> 00:03:04,000 In the first round of PCR the temperature is raised to a point at which the DNA is melted 36 00:03:04,000 --> 00:03:08,000 and the complementary strands separate from one another. 37 00:03:08,000 --> 00:03:14,000 The temperature is then lowered to a level at which the complementary strands can re-associate. 38 00:03:14,000 --> 00:03:24,000 However, since the primers are present in the mixture at huge numbers, they are most 39 00:03:24,000 --> 00:03:28,000 likely to bind at the complementary sites when the strands re-associate. 40 00:03:28,000 --> 00:03:37,000 As the temperature is lowered further, the polymerase finds the free prime ends of the 41 00:03:37,000 --> 00:03:44,000 primers and the enzyme begins to add nucleotides to the end of the primer using the complementary 42 00:03:44,000 --> 00:03:45,000 strand as a template. 43 00:03:45,000 --> 00:03:52,000 The same process occurs when DNA replicates in normal cell division. 44 00:03:52,000 --> 00:04:00,000 At the end of round one of PCR there will be two copies of the target sequence for every 45 00:04:00,000 --> 00:04:04,000 one that was present in the clinical sample. 46 00:04:04,000 --> 00:04:12,000 You can keep track of the amplification in the panel that will appear on the lower left. 47 00:04:12,000 --> 00:04:17,000 The same process is repeated in the second round of PCR. 48 00:04:17,000 --> 00:04:25,000 The theromcycler dramatically heats the sample to separate the complementary strands of DNA, 49 00:04:25,000 --> 00:04:28,000 including those that have just been synthesized. 50 00:04:28,000 --> 00:04:35,000 The temperature is lowered to allow primers to bind at their specific sites and to prime 51 00:04:35,000 --> 00:04:41,000 synthesis of complementary strands by taq polymerase when the temperature is lowered 52 00:04:41,000 --> 00:04:47,000 again. 53 00:04:47,000 --> 00:04:54,000 In the third round the same cycling of the reaction temperature occurs with melting of 54 00:04:54,000 --> 00:05:00,000 the strands, binding of primers when the temperature is lowered, and new strand synthesis when 55 00:05:00,000 --> 00:05:06,000 the strands are primed for DNA polymerase to begin adding nucleotides. 56 00:05:06,000 --> 00:05:14,000 At the end of round three there are now eight double strand copies of the target sequence 57 00:05:14,000 --> 00:05:18,000 where there was originally only one. 58 00:05:18,000 --> 00:05:25,000 The enlarging frame from the lower left will now show what happens with successive cycles 59 00:05:25,000 --> 00:05:28,000 of PCR. 60 00:05:28,000 --> 00:05:34,000 With each cycle the number of copies of the target sequence doubles so there will be sixteen 61 00:05:34,000 --> 00:05:42,000 copies after four cycles, thirty-two copies after five cycles, and sixty-four copies after 62 00:05:42,000 --> 00:05:44,000 six cycles. 63 00:05:44,000 --> 00:05:50,000 By the time the thermocycler has completed forty cycles the primers and nucleotides will 64 00:05:50,000 --> 00:05:57,000 likely be exhausted but there will theoretically be ten to the twelfth (10 ^ 12) copies. 65 00:05:57,000 --> 00:06:03,000 The target sequence will have been amplified a trillion times. 66 00:06:03,000 --> 00:06:11,000 This level of amplification produces enough of the specific DNA that it can now be visualized 67 00:06:11,000 --> 00:06:14,000 by gel electrophoresis. 68 00:06:14,000 --> 00:06:22,000 The large smear of DNA at the top of the gel represents the complex DNA that was present 69 00:06:22,000 --> 00:06:24,000 in the clinical sample. 70 00:06:24,000 --> 00:06:34,000 However, a new smaller band appears in samples taken from the later cycles of PCR. 71 00:06:34,000 --> 00:06:43,000 For diagnostic laboratory purposes the amplified DNA can be detected and quantified by more 72 00:06:43,000 --> 00:06:49,000 efficient and simpler methods than gel electrophoresis. 73 00:06:49,000 --> 00:06:53,000 One of these methods is discussed in an accompanying program.