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History of Real-Time PCR

Higuchi et al.1,2 pioneered the analysis of PCR kinetics by constructing a system that detects PCR products as
they accumulate. This “real-time” system includes the intercalator ethidium bromide in each amplification reaction,
an adapted thermal cycler to irradiate the samples with ultraviolet light, and detection of the resulting fluorescence
with a computer-controlled cooled CCD camera. Amplification produces increasing amounts of double-stranded
DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the increase in fluorescence
versus cycle number, the system produces amplification plots that provide a more complete picture of the
PCR process than assaying product accumulation after a fixed number of cycles.

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References for Real-Time PCR

1. Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences.
Biotechnology 10:413–417.

2. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions.
Biotechnology 11:1026–1030.

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