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PCR Principles

PCR principles
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).
 
 
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:
 
 
Combining reverse transcription and PCR into the RT-PCR technique brought the benefits of PCR to analysis of RNA.
 
Using primers containing sequences that were not completely complementary to the template turned PCR into a tool for in vitro mutagenesis.
 
Replacing a single polymerase with a blend of a thermostable polymerase (Taq DNA Polymerase) and a proofreading polymerase (Pwo DNA Polymerase) made PCR an indispensable tool in the analysis and mapping of entire genomes by:
  • Extending the length of the sequence that could be amplified
  • Increasing the amount of PCR product
  • Providing higher fidelity during PCR
Using a “Hot Start” approach minimized the formation of primer-dimers during PCR.
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)].
 
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.
 
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.
 
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.
 
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).
 
Combining online detection and continuous fluorescence monitoring (kinetic PCR) allowed more rapid quantification of PCR products (e.g. with LightCycler).
 
 

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