Quantitative PCR
- 18 September 2000
- journal article
- review article
- Published by Walter de Gruyter GmbH in cclm
- Vol. 38 (9), 833-836
- https://doi.org/10.1515/cclm.2000.120
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.Keywords
This publication has 18 references indexed in Scilit:
- The Biopesticide Paenibacillus popilliae Has a Vancomycin Resistance Gene Cluster Homologous to the Enterococcal VanA Vancomycin Resistance Gene ClusterAntimicrobial Agents and Chemotherapy, 2000
- Amplification of c-myc in Hepatocellular Carcinoma: Correlation with Clinicopathologic Features, Proliferative Activity and p53 OverexpressionOncology, 1999
- Amplicor HIV Monitor, NASBA HIV-1 RNA QT and Quantiplex HIV RNA version 2.0 viral load assays: a Canadian evaluationJournal of Clinical Virology, 1998
- DNA Gyrase and Topoisomerase IV Are Dual Targets of Clinafloxacin Action in Streptococcus pneumoniaeAntimicrobial Agents and Chemotherapy, 1998
- Identification of S, F1C and three PapG fimbrial adhesins in uropathogenicEscherichia coliby polymerase chain reactionFEMS Immunology & Medical Microbiology, 1998
- Mechanisms of Quinolone Resistance in Clinical Strains ofPseudomonas aeruginosaMicrobial Drug Resistance, 1998
- K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancerGastroenterology, 1997
- Detection of urovirulence factors inEscherichia coliby multiplex polymerase chain reactionFEMS Immunology & Medical Microbiology, 1995
- The impact of the PCR plateau phase on quantitative PCRBiochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
- Quantitative Polymerase Chain Reaction with Oligodeoxynucleotide Ligation Assay/Enzyme-Linked Immunosorbent Assay DetectionAnalytical Biochemistry, 1993