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In April, 1983, Kary Mullis took a drive on a
moonlit California mountain road and changed the course of molecular
biology. During that drive, he conceived the Polymerase Chain
Reaction (PCR). |
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As is shown in the figure, the reaction uses two
oligonucleotide primers that hybridize to opposite strands and flank
the target DNA sequence that is to be amplified. The elongation of
the primers is catalyzed by a heat-stable DNA polymerase (such as
Taq DNA Polymerase). A repetitive series of cycles involving
template denaturation, primer annealing, and extension of the
annealed primers by the polymerase results in exponential
accumulation of a specific DNA fragment. The ends of the fragment
are defined by the 5' ends of the primers. Because the primer
extension products synthesized in a given cycle can serve as a
template in the next cycle, the number of target DNA copies
approximately doubles every cycle; thus, 20 cycles of PCR yield
about a million copies of the target DNA.
During the last decade, innovative researchers
have continually updated the definition of "PCR applications",
increasing the usefulness and scope of the technique. For instance:
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Combining reverse transcription and PCR into
the RT-PCR technique brought the benefits of PCR to analysis
of RNA. |
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Using primers containing sequences that were
not completely complementary to the template turned PCR into a
tool for in vitro mutagenesis. |
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Replacing a single polymerase with a blend
of a thermostable polymerase (Taq DNA Polymerase) and a
proofreading polymerase (Tgo DNA Polymerase) made PCR an
indispensable tool in the analysis and mapping of entire
genomes by:
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Extending the length of the sequence that
could be amplified
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Increasing the amount of PCR product
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Providing higher fidelity during PCR
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Using a “Hot Start” approach minimized the
formation of primer-dimers during
PCR. |
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Using short primers to produce a genomic
“fingerprint” allowed analysis of organisms in which genomic
sequences are largely unknown [e.g. Differential Display,
Random Amplified Polymorphic DNA
(RAPD)]. |
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Introducing molecular “tags”, such as
digoxigenin (DIG) or biotin-labeled dUTP into the PCR product,
as it was amplified made PCR an invaluable tool for medical
diagnostics. Such labeled PCR products may either be used as
hybridization probes or be detected by use of capture probes.
For instance, with PCR-generated DIG-labeled hybridization
probes, it was possible to detect and quantify minute amounts
of a pathogen. |
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Combining in situ hybridization with PCR (in
situ PCR) made possible the localization of single nucleic
acid sequences on one chromosome within an eukaryotic
organism. |
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Extending PCR to the amplification of more
than one sequence at a time (multiplex PCR) made it possible
to compare two or more complex genomes, for instance to detect
chromosomal imbalances. |
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The first automated system that combines
amplification and detection of PCR and thereby minimizes
handling time. A true diagnostic walk away system which
guarantees integrity of the clinical results (COBAS
AMPLICOR). |
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Combining online detection and continuous
fluorescence monitoring (kinetic PCR) allowed more rapid
quantification of PCR products (e.g. with
LightCycler). |
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To learn more about the principles of PCR
and recent developments, please follow these
links: | |
