polymerase chain reaction
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Polymerase Chain Reaction. Mrs. Stewart Medical Interventions. Polymerase Chain Reaction. a lab technique that produces numerous copies of a specific segment of our DNA in a relatively short period of time. three -step process repeated over and over - PowerPoint PPT PresentationTRANSCRIPT
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Polymerase Chain Reaction
Mrs. StewartMedical Interventions
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Polymerase Chain Reaction
a lab technique that produces numerous copies of a specific segment of our DNA in a relatively short period of time. three-step processrepeated over and overproduces identical copies of the target
sequence.
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Kary Mullis1983 – Mullis
and his colleagues invented the PCR technique
Nobel Prize in 1993 
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PCR components
DNA (Taq) polymeras
e
Primers Nucleotides
Target DNA
sequence
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Taq PolymeraseThe most widely used polymerase is
that from Thermus aquaticus (Taq) – Thermophilic bacteria
Thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C
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3 steps in a PCR1.Denature2.Anneal3.Extension
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DenatureThe DNA is heated to 95oC, causing
the double stranded DNA to denature by breaking the hydrogen bonds between the strands.
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Denaturation
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AnnealThe temperature of the sample is
lowered to between 32-72oC, causing the primers to hybridize or "anneal" to their complementary sequences on either side of the target sequence.
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Anneal
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ExtensionThe temperature of the sample is
heated to between 72-75oC, which is the optimal temperature for the Taq polymerase enzyme to function.
Taq polymerase binds and extends a complementary DNA strand from each primer (adding approximately 60 bases per second, using the free-floating nucleotides)
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Extension
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As amplification proceeds, the DNA sequence between primers doubles after each cycles
(The amplification of the target sequence proceeding in an exponential fashion ( 1 2 4 8 16................) up to million of times the starting amount until enough is present to be seen by gel electrophoresis.
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How many cycles?Most PCRs should include only 25 –
35 cycles. Depends on the amount of
starting material
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Advantages of PCRUseful, non-invasive procedureSimplicity of the procedureSensitivity of the PCR
Disadvantages of PCRFalse positive results (cross contamination). False negative results (e.g. rare of
circulating fetal cells).