Quantitative analysis of gene expression "real-time PCR": Scientific letter

Abstract

Real-time quantitative PCR is the current method of choice for quantifying nucleic acids. Other descriptions of this technology include "kinetic PCR" and "homogeneous PCR". In real-time PCR, the amount of product formed is monitored during the course of the reaction by monitoring the amount of fluorescence from stains or the amount of probes introduced into the reaction, that increase proportional to the amount of product formed and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. The simplest method involves the detection of DNA-binding fluorescent stain, such as SYBR Green. This stain binds to any double-stranded DNA in the reaction. Although SYBR Green detection works very well, it may give rise to fluorescence signal in the presence of any double stranded DNA including primer-dimers product. The other three methods rely on the hybridization of fluorescence-labeled probes to the correct amplicon. These include real-time amplicon detection technologies such as Taqman or hydrolysis probes, molecular beacon technology and hybridization probes. Real-time technology has a number of advantages. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis. analysis of chromosome aberrations, and most recently protein detection by real-time immuno PCR. There are currently several real-time thermocyclers on the market. The main differences between them are the excitation and emission wavelengths that are available, speed, and the number of reactions that can be run in parallel. The importance of real-time PCR is increasing in various fields of investigation. The major reason of choice is that it is one of the most sensitive, efficient, rapid, and reproducible methods of measuring gene expression.