Caister Academic Press

Principles of the Real-time Polymerase Chain Reaction

Stephen A Bustin, Sara Zaccara and Tania Nolan
from: Polymerase Chain Reaction: Theory and Technology (Author: Mark A. Behlke, Kornelia Berghof-Jäger, Tom Brown, et al.). Caister Academic Press, U.K. (2019) Pages: 19-42.

Abstract

The real-time fluorescence-based quantitative polymerase chain reaction (qPCR) has become the benchmark technology for the detection of nucleic acids in every area of microbiology, biomedical research, biotechnology and in forensic applications. Unlike conventional (legacy) PCR, which is a qualitative end-point assay, qPCR allows accurate quantification of amplified DNA in real time during the exponential phase of the reaction. The cost of instruments and reagents is well within reach of individual laboratories, assays are easy to perform, capable of high throughput and combine high sensitivity with reliable specificity. It is possible to achieve accurate and biologically meaningful quantification if meticulous attention is paid to the details of every step of the qPCR assay, starting with sample selection, acquisition and handling through assay design, validation and optimisation. The growing awareness of the need for standardisation, quality control and the significant problems associated with inadequate reporting of the assay has resulted in the publication of guidelines for minimum information for the publication of qPCR experiments (MIQE) read more ...
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