[Script Info]
Title: 
[Events]
Format: Layer, Start, End, Style, Name, MarginL, MarginR, MarginV, Effect, Text
Dialogue: 0,0:00:00.00,0:00:02.00,Default,,0000,0000,0000,,(English captions by Andrea Matsumoto, University of Michigan.)
Dialogue: 0,0:00:02.00,0:00:10.00,Default,,0000,0000,0000,,The polymerase chain reaction or PCR can target and amplify any specific nucleic acid from
Dialogue: 0,0:00:10.00,0:00:13.00,Default,,0000,0000,0000,,complex biological samples.
Dialogue: 0,0:00:13.00,0:00:18.00,Default,,0000,0000,0000,,The procedure can be used for diagnosis to\Ndetermine whether a clinical sample contains
Dialogue: 0,0:00:18.00,0:00:23.00,Default,,0000,0000,0000,,a nuclear sequence that is known to occur\Nonly in a specific pathogen.
Dialogue: 0,0:00:23.00,0:00:31.00,Default,,0000,0000,0000,,Or the laboratory scientists may use PCR to\Namplify and color large quantities of a specific
Dialogue: 0,0:00:31.00,0:00:34.00,Default,,0000,0000,0000,,gene for research.
Dialogue: 0,0:00:34.00,0:00:39.00,Default,,0000,0000,0000,,To preform PCR you must already know the sequence\Nof the nucleic acid you wish to amplify.
Dialogue: 0,0:00:39.00,0:00:45.00,Default,,0000,0000,0000,,Then you define the boundaries of the target\Nsequence by identifying short sequences at
Dialogue: 0,0:00:45.00,0:00:48.00,Default,,0000,0000,0000,,each end on opposite strands.
Dialogue: 0,0:00:48.00,0:00:54.00,Default,,0000,0000,0000,,Here, the boundaries of the target sequence\Nare indicated by violet and green highlighting.
Dialogue: 0,0:00:54.00,0:01:00.00,Default,,0000,0000,0000,,If you move from these sequence in the five\Nprime to three prime direction, the direction
Dialogue: 0,0:01:00.00,0:01:06.00,Default,,0000,0000,0000,,of normal DNA synthesis, the violet highlighting\Nextends along one strand and the green highlighting
Dialogue: 0,0:01:06.00,0:01:09.00,Default,,0000,0000,0000,,extends along the complementary strand.
Dialogue: 0,0:01:09.00,0:01:15.00,Default,,0000,0000,0000,,It is difficult to show how PCR works using\Nthis double helix representation of DNA so
Dialogue: 0,0:01:15.00,0:01:22.00,Default,,0000,0000,0000,,the diagram with be converted to more easily\Nunderstood ladder image of the DNA.
Dialogue: 0,0:01:22.00,0:01:27.00,Default,,0000,0000,0000,,In addition to the clinical sample, the PCR\Nreaction requires three ingredients.
Dialogue: 0,0:01:27.00,0:01:33.00,Default,,0000,0000,0000,,First, there must be a massive supply of each\Nof the four nucleotides.
Dialogue: 0,0:01:33.00,0:01:40.00,Default,,0000,0000,0000,,Second, the user must add a large supply of\Nsmall synthetic primers that are designed
Dialogue: 0,0:01:40.00,0:01:47.00,Default,,0000,0000,0000,,to hybridize to the bonding sequence of either\Nend of the targeted DNA.
Dialogue: 0,0:01:47.00,0:01:53.00,Default,,0000,0000,0000,,The primers are the ingredients that make\Nthe reaction specific since only DNA that
Dialogue: 0,0:01:53.00,0:01:58.00,Default,,0000,0000,0000,,lies between these two primers will be synthesized\Nin the PCR reaction.
Dialogue: 0,0:01:58.00,0:02:04.00,Default,,0000,0000,0000,,Third, the reaction requires a DNA polymerase\Nenzyme.
Dialogue: 0,0:02:04.00,0:02:10.00,Default,,0000,0000,0000,,For PCR the polymerase is actually from a\Nbacteria that normally grows in the sea around
Dialogue: 0,0:02:10.00,0:02:14.00,Default,,0000,0000,0000,,hot geothermal vents on the ocean floor.
Dialogue: 0,0:02:14.00,0:02:20.00,Default,,0000,0000,0000,,The bacterium is called Thermus Aquaticus\Nand the polymerase is called Taq polymerase
Dialogue: 0,0:02:20.00,0:02:21.00,Default,,0000,0000,0000,,for short.
Dialogue: 0,0:02:21.00,0:02:29.00,Default,,0000,0000,0000,,This exotic enzyme is used because it is not\Ninactivated by the high temperatures generated
Dialogue: 0,0:02:29.00,0:02:31.00,Default,,0000,0000,0000,,in the PCR reaction.
Dialogue: 0,0:02:31.00,0:02:38.00,Default,,0000,0000,0000,,All these elements are mixed together in appropriate\Nproportions and placed in an instrument called
Dialogue: 0,0:02:38.00,0:02:40.00,Default,,0000,0000,0000,,a thermocycler.
Dialogue: 0,0:02:40.00,0:02:47.00,Default,,0000,0000,0000,,This instrument can be programed to change\Nthe temperature of the mixture through a series
Dialogue: 0,0:02:47.00,0:02:49.00,Default,,0000,0000,0000,,of repetitive cycles.
Dialogue: 0,0:02:49.00,0:02:57.00,Default,,0000,0000,0000,,The temperature of the reaction in this demonstration\Nis presented in the lower right panel.
Dialogue: 0,0:02:57.00,0:03:04.00,Default,,0000,0000,0000,,In the first round of PCR the temperature\Nis raised to a point at which the DNA is melted
Dialogue: 0,0:03:04.00,0:03:08.00,Default,,0000,0000,0000,,and the complementary strands separate from\None another.
Dialogue: 0,0:03:08.00,0:03:14.00,Default,,0000,0000,0000,,The temperature is then lowered to a level\Nat which the complementary strands can re-associate.
Dialogue: 0,0:03:14.00,0:03:24.00,Default,,0000,0000,0000,,However, since the primers are present in\Nthe mixture at huge numbers, they are most
Dialogue: 0,0:03:24.00,0:03:28.00,Default,,0000,0000,0000,,likely to bind at the complementary sites\Nwhen the strands re-associate.
Dialogue: 0,0:03:28.00,0:03:37.00,Default,,0000,0000,0000,,As the temperature is lowered further, the\Npolymerase finds the free prime ends of the
Dialogue: 0,0:03:37.00,0:03:44.00,Default,,0000,0000,0000,,primers and the enzyme begins to add nucleotides\Nto the end of the primer using the complementary
Dialogue: 0,0:03:44.00,0:03:45.00,Default,,0000,0000,0000,,strand as a template.
Dialogue: 0,0:03:45.00,0:03:52.00,Default,,0000,0000,0000,,The same process occurs when DNA replicates\Nin normal cell division.
Dialogue: 0,0:03:52.00,0:04:00.00,Default,,0000,0000,0000,,At the end of round one of PCR there will\Nbe two copies of the target sequence for every
Dialogue: 0,0:04:00.00,0:04:04.00,Default,,0000,0000,0000,,one that was present in the clinical sample.
Dialogue: 0,0:04:04.00,0:04:12.00,Default,,0000,0000,0000,,You can keep track of the amplification in\Nthe panel that will appear on the lower left.
Dialogue: 0,0:04:12.00,0:04:17.00,Default,,0000,0000,0000,,The same process is repeated in the second\Nround of PCR.
Dialogue: 0,0:04:17.00,0:04:25.00,Default,,0000,0000,0000,,The theromcycler dramatically heats the sample\Nto separate the complementary strands of DNA,
Dialogue: 0,0:04:25.00,0:04:28.00,Default,,0000,0000,0000,,including those that have just been synthesized.
Dialogue: 0,0:04:28.00,0:04:35.00,Default,,0000,0000,0000,,The temperature is lowered to allow primers\Nto bind at their specific sites and to prime
Dialogue: 0,0:04:35.00,0:04:41.00,Default,,0000,0000,0000,,synthesis of complementary strands by taq\Npolymerase when the temperature is lowered
Dialogue: 0,0:04:41.00,0:04:47.00,Default,,0000,0000,0000,,again.
Dialogue: 0,0:04:47.00,0:04:54.00,Default,,0000,0000,0000,,In the third round the same cycling of the\Nreaction temperature occurs with melting of
Dialogue: 0,0:04:54.00,0:05:00.00,Default,,0000,0000,0000,,the strands, binding of primers when the temperature\Nis lowered, and new strand synthesis when
Dialogue: 0,0:05:00.00,0:05:06.00,Default,,0000,0000,0000,,the strands are primed for DNA polymerase\Nto begin adding nucleotides.
Dialogue: 0,0:05:06.00,0:05:14.00,Default,,0000,0000,0000,,At the end of round three there are now eight\Ndouble strand copies of the target sequence
Dialogue: 0,0:05:14.00,0:05:18.00,Default,,0000,0000,0000,,where there was originally only one.
Dialogue: 0,0:05:18.00,0:05:25.00,Default,,0000,0000,0000,,The enlarging frame from the lower left will\Nnow show what happens with successive cycles
Dialogue: 0,0:05:25.00,0:05:28.00,Default,,0000,0000,0000,,of PCR.
Dialogue: 0,0:05:28.00,0:05:34.00,Default,,0000,0000,0000,,With each cycle the number of copies of the\Ntarget sequence doubles so there will be sixteen
Dialogue: 0,0:05:34.00,0:05:42.00,Default,,0000,0000,0000,,copies after four cycles, thirty-two copies\Nafter five cycles, and sixty-four copies after
Dialogue: 0,0:05:42.00,0:05:44.00,Default,,0000,0000,0000,,six cycles.
Dialogue: 0,0:05:44.00,0:05:50.00,Default,,0000,0000,0000,,By the time the thermocycler has completed\Nforty cycles the primers and nucleotides will
Dialogue: 0,0:05:50.00,0:05:57.00,Default,,0000,0000,0000,,likely be exhausted but there will theoretically\Nbe ten to the twelfth (10 ^ 12) copies.
Dialogue: 0,0:05:57.00,0:06:03.00,Default,,0000,0000,0000,,The target sequence will have been amplified\Na trillion times.
Dialogue: 0,0:06:03.00,0:06:11.00,Default,,0000,0000,0000,,This level of amplification produces enough\Nof the specific DNA that it can now be visualized
Dialogue: 0,0:06:11.00,0:06:14.00,Default,,0000,0000,0000,,by gel electrophoresis.
Dialogue: 0,0:06:14.00,0:06:22.00,Default,,0000,0000,0000,,The large smear of DNA at the top of the gel\Nrepresents the complex DNA that was present
Dialogue: 0,0:06:22.00,0:06:24.00,Default,,0000,0000,0000,,in the clinical sample.
Dialogue: 0,0:06:24.00,0:06:34.00,Default,,0000,0000,0000,,However, a new smaller band appears in samples\Ntaken from the later cycles of PCR.
Dialogue: 0,0:06:34.00,0:06:43.00,Default,,0000,0000,0000,,For diagnostic laboratory purposes the amplified\NDNA can be detected and quantified by more
Dialogue: 0,0:06:43.00,0:06:49.00,Default,,0000,0000,0000,,efficient and simpler methods than gel electrophoresis.
Dialogue: 0,0:06:49.00,0:06:53.00,Default,,0000,0000,0000,,One of these methods is discussed in an accompanying\Nprogram.