Developments in quantitative PCR.
In recent years the growing interest in quantitative applications of the polymerase chain reaction (PCR) has favoured the development of a large number of assay procedures suitable for this purpose. In this paper we review some basic principles of quantitative PCR and in particular the role of reference materials and calibrators and the different strategies adopted for nucleic acid quantification. We focus on two methodological approaches for quantitative PCR in this review: competitive PCR and real-time quantitative PCR based on the use of fluorogenic probes. The first is one of the most common methods of quantitative PCR and we discuss the structure of the competitors and the various assay procedures. The second section is dedicated to a recent promising technology for quantitative PCR in which the use of fluorogenic probes and dedicated instrumentation allows the development of homogeneous methods. Assay performance of these methods in terms of practicability and reliability indicates that these kinds of technologies will have a widespread use in the clinical laboratory in the near future.
Department of Clinical Pathophysiology, University of Florence, Italy.
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C
Orlando
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Pinzani
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Pazzagli
D000818:Animals; D001667:Binding, Competitive; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000592:standards; D005456:Fluorescent Dyes; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000592:standards; D012015:Reference Standards
D005456:Fluorescent Dyes; D012313:RNA; D004247:DNA