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(English captions by Andrea Matsumoto, University of Michigan.)

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The polymerase chain reaction or PCR can target and amplify any specific nucleic acid from

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complex biological samples.

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The procedure can be used for diagnosis to[br]determine whether a clinical sample contains

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a nuclear sequence that is known to occur[br]only in a specific pathogen.

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Or the laboratory scientists may use PCR to[br]amplify and color large quantities of a specific

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gene for research.

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To preform PCR you must already know the sequence[br]of the nucleic acid you wish to amplify.

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Then you define the boundaries of the target[br]sequence by identifying short sequences at

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each end on opposite strands.

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Here, the boundaries of the target sequence[br]are indicated by violet and green highlighting.

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If you move from these sequence in the five[br]prime to three prime direction, the direction

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of normal DNA synthesis, the violet highlighting[br]extends along one strand and the green highlighting

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extends along the complementary strand.

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It is difficult to show how PCR works using[br]this double helix representation of DNA so

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the diagram with be converted to more easily[br]understood ladder image of the DNA.

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In addition to the clinical sample, the PCR[br]reaction requires three ingredients.

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First, there must be a massive supply of each[br]of the four nucleotides.

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Second, the user must add a large supply of[br]small synthetic primers that are designed

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to hybridize to the bonding sequence of either[br]end of the targeted DNA.

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The primers are the ingredients that make[br]the reaction specific since only DNA that

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lies between these two primers will be synthesized[br]in the PCR reaction.

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Third, the reaction requires a DNA polymerase[br]enzyme.

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For PCR the polymerase is actually from a[br]bacteria that normally grows in the sea around

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hot geothermal vents on the ocean floor.

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The bacterium is called Thermus Aquaticus[br]and the polymerase is called Taq polymerase

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for short.

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This exotic enzyme is used because it is not[br]inactivated by the high temperatures generated

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in the PCR reaction.

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All these elements are mixed together in appropriate[br]proportions and placed in an instrument called

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a thermocycler.

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This instrument can be programed to change[br]the temperature of the mixture through a series

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of repetitive cycles.

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The temperature of the reaction in this demonstration[br]is presented in the lower right panel.

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In the first round of PCR the temperature[br]is raised to a point at which the DNA is melted

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and the complementary strands separate from[br]one another.

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The temperature is then lowered to a level[br]at which the complementary strands can re-associate.

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However, since the primers are present in[br]the mixture at huge numbers, they are most

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likely to bind at the complementary sites[br]when the strands re-associate.

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As the temperature is lowered further, the[br]polymerase finds the free prime ends of the

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primers and the enzyme begins to add nucleotides[br]to the end of the primer using the complementary

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strand as a template.

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The same process occurs when DNA replicates[br]in normal cell division.

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At the end of round one of PCR there will[br]be two copies of the target sequence for every

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one that was present in the clinical sample.

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You can keep track of the amplification in[br]the panel that will appear on the lower left.

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The same process is repeated in the second[br]round of PCR.

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The theromcycler dramatically heats the sample[br]to separate the complementary strands of DNA,

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including those that have just been synthesized.

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The temperature is lowered to allow primers[br]to bind at their specific sites and to prime

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synthesis of complementary strands by taq[br]polymerase when the temperature is lowered

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again.

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In the third round the same cycling of the[br]reaction temperature occurs with melting of

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the strands, binding of primers when the temperature[br]is lowered, and new strand synthesis when

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the strands are primed for DNA polymerase[br]to begin adding nucleotides.

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At the end of round three there are now eight[br]double strand copies of the target sequence

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where there was originally only one.

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The enlarging frame from the lower left will[br]now show what happens with successive cycles

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of PCR.

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With each cycle the number of copies of the[br]target sequence doubles so there will be sixteen

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copies after four cycles, thirty-two copies[br]after five cycles, and sixty-four copies after

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six cycles.

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By the time the thermocycler has completed[br]forty cycles the primers and nucleotides will

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likely be exhausted but there will theoretically[br]be ten to the twelfth (10 ^ 12) copies.

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The target sequence will have been amplified[br]a trillion times.

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This level of amplification produces enough[br]of the specific DNA that it can now be visualized

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by gel electrophoresis.

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The large smear of DNA at the top of the gel[br]represents the complex DNA that was present

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in the clinical sample.

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However, a new smaller band appears in samples[br]taken from the later cycles of PCR.

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For diagnostic laboratory purposes the amplified[br]DNA can be detected and quantified by more

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efficient and simpler methods than gel electrophoresis.

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One of these methods is discussed in an accompanying[br]program.