[8] Quantitative PCR: Analysis of rare mitochondrial DNA mutations in central nervous system tissues

Publisher Summary This chapter discusses the use of polymerase chain reaction (PCR) in quantitative applications. Although the sensitivity of PCR has rendered it the method of choice in the detection of low amounts of nucleic acid target, its utility as a quantitative tool has been slower in gaining credibility. The ability of PCR to make rare sequences detectable also makes it extremely challenging to extrapolate the amounts of initial input template molecules from the measured amounts of product molecules after many cycles of amplification. In the past few years, a number of PCR strategies based on different methodological and technical approaches have been adopted for the semiquantitative or quantitative analysis of nucleic acids. These strategies deal with the basic problem of making the amplification process as predictable as possible. Because of the exponential nature of PCR, even minor variations in efficiency can lead to large deviations at the end of the reaction that will confound the quantitative relationship between the product and initial template. An understanding of the theoretical and technical aspects of the kinetics of PCR product accumulation is imperative to designing quantitative PCR experiments.