Polymerase Chain Reaction Mrs. Stewart Medical Interventions.
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Polymerase Chain Reaction
Mrs. StewartMedical 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 processrepeated over and overproduces identical copies of the target
sequence.
Kary Mullis
1983 – Mullis and his colleagues invented the PCR technique
Nobel Prize in 1993 
PCR components
DNA (Taq) polymeras
e
Primers Nucleotides
Target DNA
sequence
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
3 steps in a PCR
1.Denature
2.Anneal
3.Extension
DenatureThe DNA is heated to 95oC, causing
the double stranded DNA to denature by breaking the hydrogen bonds between the strands.
Denaturation
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.
Anneal
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)
Extension
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.
How many cycles?Most PCRs should include only 25 –
35 cycles. Depends on the amount of
starting material
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).
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