Primers and Probes

Abstract

Nucleic acid probes are the crucial reagents to detect specific nucleic acid sequences by means of hybridisation. Visualisation of a specific hybridised probe, either in solution or attached to a solid phase, is the basic principle of a hybridisation test. A large number of enzymatic and organic chemical reagents and methods have been developed for the detection of probe molecules. Probes can be labelled with fluorescent dyes, haptens or enzymes. Detection can occur through autoradiography, chemoluminescence, fluorescence or enzymatic substrate conversion. Primers hybridising to complementary sequences are essential for multiple molecular techniques that require DNA and/or RNA synthesis. Primers provide a double-stranded 3-OH terminus acting as a starting point for a variety of molecular strategies. The most important application of primers is in the PCR reaction. In a number of PCR variants, both primers and probes are needed. To visualise the level of amplification, fluorescence is measured in a quantitative way. Next to PCR, primers are used in cDNA synthesis, sequencing and probe labelling. The oligonucleotide sequence of primers and probes should be checked for uniqueness and for physical–chemical characteristics. Design and control of primers and probes using special software and (Web-based) applications is a (daily) part of molecular diagnostics, especially when developing new diagnostic assays.