Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a rapid procedure for in vitro enzymatic amplification of specific segments of DNA. Amplification of the genes of interest occurs by selecting and synthesizing "primers"—stretches of DNA that span the region of interest to be "filled in"—and by heating DNA to make it single stranded, allowing polymerase and primers to bind. The polymerase then reads the "blank" stretch of DNA between the primers and synthesizes DNA corresponding to the region of interest. This is usually done in "thermal cyclers" that heat the DNA at regular intervals and allow "cycles" of PCR to amplify the genes repetitively. To avoid continual addition of polymerase, the DNA polymerase used is often from bacteria that inhabit hot springs or hot ocean vents. This polymerase is not denatured by heating and can therefore support several cycles of PCR.

A variation on PCR is reverse transcriptase PCR (or RT-PCR), in which the RNA is the template. Reverse transcriptase (the "RT" of RT-PCR) is employed to synthesize cDNA from the RNA. Enzymatic amplification of this cDNA is then accomplished with PCR.

Real-time PCR is based on the method of RT-PCR, following the reverse transcription of RNA into cDNA. Real-time PCR requires suitable detection chemistries to report the presence of PCR products, as well as an instrument to monitor the amplification in real time through recording of the change in fluorescence (Wittwer et al. 1997). Generally, chemistries in PCR consist of fluorescent probes. Several probes exist, including DNA-binding dyes like EtBr or SYBR green I, hydrolysis probes (5'-nuclease probes), and hybridization probes (Valasek and Repa 2005). Each type of probe has its own unique characteristics, but the strategy for each is to link a change in fluorescence to amplification of DNA (Kubista et al. 2006; Lind et al. 2006). The instrumentation to detect the production of PCR must be able to input energy for excitation of fluorescent chemistries at the desired wavelength and simultaneously detect a particular emission wavelength. Many instrument platforms are available for real-time PCR. The major differences among them are the excitation and emission wavelengths that are available, speed, and the number of reactions that can be run in parallel (Kubista 2004).

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