Review
The real-time polymerase chain reaction

https://doi.org/10.1016/j.mam.2005.12.007Get rights and content

Abstract

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A–Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year’s Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413–417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.

Section snippets

PCR amplification

PCR is performed on a DNA template, which can be single or double-stranded. Also needed are two oligonucleotide primers that flank the DNA sequence to be amplified, dNTPs, which are the four nucleotide triphosphates, a heat-stable polymerase, and magnesium ions in the buffer. The reaction is performed by temperature cycling. High temperature is applied to separate (melt) the strands of the double helical DNA, then temperature is lowered to let primers anneal to the template, and finally the

Gene expression measurements

Before a gene expression measurement can be performed by real-time PCR, the mRNA in the sample must be copied to cDNA by reverse transcription (RT) (Fig. 8). The RT step is critical for sensitive and accurate quantification, since the amounts of cDNAs produced must correctly reflect the input amounts of the mRNAs. Comparing the technical reproducibilities of the RT reaction and the PCR it was found that the RT reaction contributes with most of the variation to the experimental determination of

Acknowledgement

This work was supported in part by project no. AVOZ50520514 awarded by the Academy of Sciences of the Czech Republic.

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