Quantitative PCR

Abstract

The classic molecular biology methods like Northern or Southern blot analyse non-amplified DNA or RNA, but need large amounts of nucleic acids, in many instances from tissues or cells that are heterogeneous. In contrast, polymerase chain reaction (PCR)-based techniques allow us to obtain genetic information through the specific amplification of nucleic acid sequences starting with a very low number of target copies. These reactions are characterized by a logarithmic amplification of the target sequences i.e. increase of PCR copies followed by a plateau phase showing a rapid decrease to zero of copy number increment per cycle. Accordingly, the amount of specific DNA product at the end of the PCR run bears no correlation to the number of target copies present in the original specimen. However, many applications in medicine or research require quantification of the number of specific targets in the specimen. This has generated a rapidly increasing need for the development of quantitative PCR techniques. Prominent examples are the determination of viral load in blood specimens for the diagnosis of HIV or HCV infections, the determination of changes in gene dosage through amplification or deletion e.g. of MDR-1, erb-B2, c-myc or the loss of heterozygosity in general. Finally, the analysis of gene expression on the mRNA level does require quantitative approaches to reverse transcriptase PCR, e.g. for studies in morphogenesis or the profiling of cancer cells. Recent advances in technology allow detection of the increment per cycle of a specifically generated PCR product in “real-time mode”. Together with the new powerful methods to dissect heterogeneous tissues or fractionate bodily fluids, this now sets the stage for a detailed analysis not only of the genes and genetic changes within a single cell, but also of the use such cell makes of its genes e.g. in pharmacogenomics. Examples of recent developments of the technology and their applications will be given.