PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Quantitative PCR and its real time dimensions by Arseni Markoff, Ph.D. Those of you who have worked in the field of quantitative PCR (qPCR) , will certainly remember the early ‘90s, when this assay was very tedious in its original form. Difficulties were 1) to make sure that the PCR was within the linear range of amplification (the fraction where the PCR signal is directly proportional to the input copy number) and 2) finding a proper method to detect the product, once linear amplification is accomplished. To ensure linearity, one had to apply serial dilutions of the template or alter the number of amplification cycles for each amplicon in question. Others would add a competitor template, perform limiting dilution assays, or use a PCR mimic with similar primers sequence to be co-amplified in the course of the PCR reaction. Detection of amplicons was accomplished electrophoretically, using radioactive labels or nonradioactive staining and corresponding DNA probes. Nowadays most of us use “real time” quantitative PCR and the old days are but a memory. In real time qPCR (real time PCR) worrying about linear amplification, running gels, working with radioactivity, and gathering numbers are a thing of the past. Generating data in 2-3 hours is now a reality. Other advantages of the real time PCR are enhanced sensitivity, greater throughput, use of a closed-tube system, standardization, running simultaneously multiplex reactions and no need for post-PCR manipulations. The technology to detect PCR products in real time has been available for more than 5 years, but in the last few years it has seen a dramatic increase in use. MedLine search using real-time and PCR as key words yields 52 citations in 1998 and 351 citations in 2003. Various manufacturers are gearing up real time PCR instrumentation and affiliated technology (software, probes, etc.) and more are likely to be released in the near future. Real time PCR is used to quantify gene expression (absolute or relative) and to quantify viral or microorganismal load in infected samples. This method is also very important for genetic applications including the allelic discrimination in various analyses including SNP detection with molecular beacons or multiplex detection of different alleles, for quantitative visulaization of DNA methylation patterns after bisulfite treatment in cancer cells and tissues or in inherited gene disorders. Real time PCR is also an important tool for pathology and forensics, for quantitative analysis of nucleic acids in archival biopsies or forensic samples, to name current important applications. Many technological advances have been made in using PCR since its discovery in the mid-1980s. While technology and methodology for real-time detection of quantitative PCR was possible prior to the development of real time PCR, it required specific training, rigorous re-testing, and lots of technical manipulations. The development of real time PCR has enabled scientists to gain knowledge on fundamental and applied questions in relatively straightforward and automated fashion. Although some of the difficulties may still need to be worked out, the foundation has been set for the use of real time quantitative PCR in routine diagnostic procedures. This rapid development within a very short period of time has led to one of those defining turns in technological development when the old protocols are well remembered but obsolete for current applications. ### Dr. Markoff has been a member of The Science Advisory Board since December, 2001 and is currently serving on the Steering Committee. ### << Previous Next >> [ View All Perspectives ] |
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