WEBVTT 00:00:00.000 --> 00:00:02.000 (English captions by Andrea Matsumoto, University of Michigan.) 00:00:02.000 --> 00:00:10.000 The polymerase chain reaction or PCR can target and amplify any specific nucleic acid from 00:00:10.000 --> 00:00:13.000 complex biological samples. 00:00:13.000 --> 00:00:18.000 The procedure can be used for diagnosis to determine whether a clinical sample contains 00:00:18.000 --> 00:00:23.000 a nuclear sequence that is known to occur only in a specific pathogen. 00:00:23.000 --> 00:00:31.000 Or the laboratory scientists may use PCR to amplify and color large quantities of a specific 00:00:31.000 --> 00:00:34.000 gene for research. 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. 00:00:39.000 --> 00:00:45.000 Then you define the boundaries of the target sequence by identifying short sequences at 00:00:45.000 --> 00:00:48.000 each end on opposite strands. 00:00:48.000 --> 00:00:54.000 Here, the boundaries of the target sequence are indicated by violet and green highlighting. 00:00:54.000 --> 00:01:00.000 If you move from these sequence in the five prime to three prime direction, the direction 00:01:00.000 --> 00:01:06.000 of normal DNA synthesis, the violet highlighting extends along one strand and the green highlighting 00:01:06.000 --> 00:01:09.000 extends along the complementary strand. 00:01:09.000 --> 00:01:15.000 It is difficult to show how PCR works using this double helix representation of DNA so 00:01:15.000 --> 00:01:22.000 the diagram with be converted to more easily understood ladder image of the DNA. 00:01:22.000 --> 00:01:27.000 In addition to the clinical sample, the PCR reaction requires three ingredients. 00:01:27.000 --> 00:01:33.000 First, there must be a massive supply of each of the four nucleotides. 00:01:33.000 --> 00:01:40.000 Second, the user must add a large supply of small synthetic primers that are designed 00:01:40.000 --> 00:01:47.000 to hybridize to the bonding sequence of either end of the targeted DNA. 00:01:47.000 --> 00:01:53.000 The primers are the ingredients that make the reaction specific since only DNA that 00:01:53.000 --> 00:01:58.000 lies between these two primers will be synthesized in the PCR reaction. 00:01:58.000 --> 00:02:04.000 Third, the reaction requires a DNA polymerase enzyme. 00:02:04.000 --> 00:02:10.000 For PCR the polymerase is actually from a bacteria that normally grows in the sea around 00:02:10.000 --> 00:02:14.000 hot geothermal vents on the ocean floor. 00:02:14.000 --> 00:02:20.000 The bacterium is called Thermus Aquaticus and the polymerase is called Taq polymerase 00:02:20.000 --> 00:02:21.000 for short. 00:02:21.000 --> 00:02:29.000 This exotic enzyme is used because it is not inactivated by the high temperatures generated 00:02:29.000 --> 00:02:31.000 in the PCR reaction. 00:02:31.000 --> 00:02:38.000 All these elements are mixed together in appropriate proportions and placed in an instrument called 00:02:38.000 --> 00:02:40.000 a thermocycler. 00:02:40.000 --> 00:02:47.000 This instrument can be programed to change the temperature of the mixture through a series 00:02:47.000 --> 00:02:49.000 of repetitive cycles. 00:02:49.000 --> 00:02:57.000 The temperature of the reaction in this demonstration is presented in the lower right panel. 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 00:03:04.000 --> 00:03:08.000 and the complementary strands separate from one another. 00:03:08.000 --> 00:03:14.000 The temperature is then lowered to a level at which the complementary strands can re-associate. 00:03:14.000 --> 00:03:24.000 However, since the primers are present in the mixture at huge numbers, they are most 00:03:24.000 --> 00:03:28.000 likely to bind at the complementary sites when the strands re-associate. 00:03:28.000 --> 00:03:37.000 As the temperature is lowered further, the polymerase finds the free prime ends of the 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 00:03:44.000 --> 00:03:45.000 strand as a template. 00:03:45.000 --> 00:03:52.000 The same process occurs when DNA replicates in normal cell division. 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 00:04:00.000 --> 00:04:04.000 one that was present in the clinical sample. 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. 00:04:12.000 --> 00:04:17.000 The same process is repeated in the second round of PCR. 00:04:17.000 --> 00:04:25.000 The theromcycler dramatically heats the sample to separate the complementary strands of DNA, 00:04:25.000 --> 00:04:28.000 including those that have just been synthesized. 00:04:28.000 --> 00:04:35.000 The temperature is lowered to allow primers to bind at their specific sites and to prime 00:04:35.000 --> 00:04:41.000 synthesis of complementary strands by taq polymerase when the temperature is lowered 00:04:41.000 --> 00:04:47.000 again. 00:04:47.000 --> 00:04:54.000 In the third round the same cycling of the reaction temperature occurs with melting of 00:04:54.000 --> 00:05:00.000 the strands, binding of primers when the temperature is lowered, and new strand synthesis when 00:05:00.000 --> 00:05:06.000 the strands are primed for DNA polymerase to begin adding nucleotides. 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 00:05:14.000 --> 00:05:18.000 where there was originally only one. 00:05:18.000 --> 00:05:25.000 The enlarging frame from the lower left will now show what happens with successive cycles 00:05:25.000 --> 00:05:28.000 of PCR. 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 00:05:34.000 --> 00:05:42.000 copies after four cycles, thirty-two copies after five cycles, and sixty-four copies after 00:05:42.000 --> 00:05:44.000 six cycles. 00:05:44.000 --> 00:05:50.000 By the time the thermocycler has completed forty cycles the primers and nucleotides will 00:05:50.000 --> 00:05:57.000 likely be exhausted but there will theoretically be ten to the twelfth (10 ^ 12) copies. 00:05:57.000 --> 00:06:03.000 The target sequence will have been amplified a trillion times. 00:06:03.000 --> 00:06:11.000 This level of amplification produces enough of the specific DNA that it can now be visualized 00:06:11.000 --> 00:06:14.000 by gel electrophoresis. 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 00:06:22.000 --> 00:06:24.000 in the clinical sample. 00:06:24.000 --> 00:06:34.000 However, a new smaller band appears in samples taken from the later cycles of PCR. 00:06:34.000 --> 00:06:43.000 For diagnostic laboratory purposes the amplified DNA can be detected and quantified by more 00:06:43.000 --> 00:06:49.000 efficient and simpler methods than gel electrophoresis. 00:06:49.000 --> 00:06:53.000 One of these methods is discussed in an accompanying program.