Principles Of Operation - Applied Biosystems GeneAmp PCR System 9600 User Manual

Principles of Operation

The GeneAmp PCR System 9600 automates the Polymerase
Chain Reaction (PCR) technique for amplifying DNA.
The Polymerase Chain Reaction (PCR) technique is conceptually
a very simple method for amplifying nucleic acids. It mimics the
natural DNA replication process in that the number of DNA
molecules generated by the Polymerase Chain Reaction doubles
after each cycle.
In the PCR technique, the typical cycle consists of three steps:
o
o
o
The anneal and extend steps can be combined into a single
setpoint, called two-temperature PCR.
The original DNA segment can be either a small, discrete
molecule whose sequence is already known or a part of a much
larger molecule in a complex mixture, such as a chromosome
fragment.
The product of the reaction will be discrete double-stranded DNA
molecules whose termini will be determined by the set of primers
used. Because the copy number of the target DNA doubles after
each cycle, amplification through 25 cycles would theoretically
yield a 33 million fold increase of specific product.
The PCR method readily lends itself to research applications.
Cloning of the amplified sequence can be facilitated by extending
the 5' end of the primers with short sequences harboring
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Denaturing the template DNA by heating it to a high
temperature (94 degrees C to 95 degrees C), thereby
producing two single strands of DNA.
Annealing of the target-specific primers to the two separated
DNA strands by cooling the reaction mixture to a lower
temperature (37 degrees C to 65 degrees C).
Extending the annealed primers with a DNA polymerase by
warming the reaction mixture to an intermediate temperature
(72 degrees C).