Quantitative PCR

The classic molecular biology methods like Northern or Southern blot analys e non-amplified DNA or RNA, but need large amounts of nucleic acids, in man y instances from tissues or cells that are heterogeneous. In contrast, poly merase chain reaction (PCR)-based techniques allow us to obtain genetic inf ormation through the specific amplification of nucleic acid sequences start ing with a very low number of target copies. These reactions are characteri zed by a logarithmic amplification of the target sequences Le. 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 pr oduct at the end of the PCR run bears no correlation to the number of targe t copies present in the original specimen. However, many applications in me dicine or research require quantification of the number of specific targets in the specimen. This has generated a rapidly increasing need for the deve lopment of quantitative PCR techniques. Prominent examples are the determin ation of viral load in blood specimens for the diagnosis of HIV or HCV infe ctions, the determination of changes in gene dosage through amplification o r deletion e.g. of MDR-1, erb-B2, c-myc or the loss of heterozygosity in ge neral. Finally, the analysis of gene expression on the mRNA level does requ ire quantitative approaches to reverse transcriptase PCR, e.g. for studies in morphogenesis or the profiling of cancer cells. Recent advances in techn ology allow detection of the increment per cycle of a specifically generate d PCR product in "real-time mode". Together with the new powerful methods t o dissect heterogeneous tissues or fractionate bodily fluids, this now sets the stage for a detailed analysis not only of the genes and genetic change s 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 a nd their applications will be given.