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Real-Time Multiplex PCR from Genomic DNA Using the iCycler iQ Detection System

Faye Boeckman, Larissa Tan, Marni Brisson, Rob Park, and Keith Hamby, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr., Hercules, CA 94547


Introduction
When researchers need to measure the amount of RNA or DNA in a preparation, they typically employ a traditional biochemical approach. High concentrations of nucleic acid can be estimated by staining agarose or acrylamide gels after electrophoresis. With less material, or for more exact determination, the concentration can be estimated by spectroscopy or fluorometry. Occasionally, the amount of nucleic acid present is so small that none of the traditional measurement methods are appropriate. In these cases, researchers have turned to the polymerase chain reaction (PCR) to make more copies of the desired nucleic acid. After amplification they measure the amount of DNA produced in the reaction and finally calculate the starting amount of nucleic acid based on post-PCR measurements. For a number of technical reasons, accurate calculation of the starting amount of nucleic acid is not possible, and even relative comparisons between 2 amplified samples are generally not valid because of differences in efficiency and specificity of the 2 amplification reactions. In gene expression studies, when the starting amount of nucleic acid is diminishingly small, or for the most demanding sensitivity, such as monitoring the number of viral particles present in a patients blood sample, real-time quantitative PCR is the only reasonable approach. In real-time quantitative PCR, a fluorescent reporter molecule is added to the chemical reaction. This reporter may be specific for the desired amplification product, or it can be nonspecific, used on
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