Submitted to: Current Status of Molecular Techniques in Aquaculture
Publication Type: Book / Chapter
Publication Acceptance Date: 11/1/2007
Publication Date: N/A
Interpretive Summary: Quantitative polymerase chain reaction analysis, or QPCR, is a method for using the exponential amplification of sequence by PCR to quantify the level of an mRNA message or DNA sequence present in a sample. This method has now become a mainstay in molecular analysis for the determination of absolute or relative gene expression or genome numbers from isolated samples. The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is has become an essential procedure in all molecular laboratories for sequence amplification and detection, genotyping, sequencing and cloning. From these origins has developed the method of using PCR for the quantification of DNA or mRNA from samples. Quantitative polymerase chain reaction (qPCR) is a modification of the polymerase chain reaction used to rapidly measure the quantity of a nucleic acid present in a sample. Like other forms of polymerase chain reaction, the process is used to amplify DNA samples, via the temperature-mediated enzyme DNA polymerase. The monitoring of accumulating amplicon in real time has been made possible by the labeling of primers, probes or amplicons with fluorogenic molecules. This chemistry has clear benefits over radiogenic oligoprobes that include an avoidance of radioactive emissions, ease of disposal and an extended shelf life.
Technical Abstract: Real-time PCR has engendered wider acceptance of quantitative PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination when compared to older methods. Also post-amplification manipulation of the amplicon is not required for quantification when using real-time PCR, therefore these assays are described as ‘closed’ or homogeneous systems. The advantages of homogeneous systems include a reduced result turnaround, minimization of the potential for carry-over contamination and the ability to closely scrutinize the assay’s performance. QPCR is used in a wide array of scientific research areas, including but not limited to- developmental biology, physiology, immunology, and a majority of other studies evaluating the genetic effect of physiological changes. Several of the reasons for the rapid increase in the use of QPCR since the late 90’s include the huge influx of genetic sequence for a multitude of different organisms, advances in DNA and RNA isolation procedures and reagents, several different platforms and chemistries are now available for running samples, (complete analysis post run without further steps or manipulation) and improved software for the generation of sequences necessary to perform QPCR. The increased speed of real-time PCR is largely due to reduced cycle times, removal of post-PCR detection procedures and the use of fluorogenic labels and sensitive methods of detecting their emissions