Stains, labels and detection strategies for nucleic acids assays

Ann Clin Biochem 2002;39:114-129
doi:10.1258/0004563021901865
© 2002 Association for Clinical Biochemistry

 

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Review Article


Larry J Kricka


Department of Pathology and Laboratory Medicine, University of Pennsylvania Philadelphia, PA 19104, USA

Selected developments and trends in stains, labels and strategies for detecting and measuring nucleic acids (DNA, RNA) and related molecules [e.g. oligo(deoxy)nucleotides, nucleic acid fragments and polymerase chain reaction products] are surveyed based on the literature in the final decade of the 20th century (1991-2000). During this period, important families of cyanine dyes were developed for sensitive detection of double-stranded DNA, single-stranded DNA, and oligo(deoxy)nucleotides in gels and in solution, and families of energy transfer primers were produced for DNA sequencing applications. The continuing quest for improved labels for hybridization assays has produced a series of candidate labels including genes encoding enzymes, microparticles (e.g. quantum dots, nanocrystals, phosphors), and new examples of the fluorophore (e.g. cyanine dyes) and enzyme class of labels (e.g. firefly luciferase mutants). Label detection technologies for use in northern and southern blotting assays have focused on luminescent methods, particularly enhanced chemiluminescence for peroxidase labels and adamantyl 1,2-dioxetanes for alkaline phosphatase labels. Sets of labels have been selected to meet the demands of multicolour assays (e.g. four-colour sequencing and single nucleotide primer extension assays). Non-separation assay formats have emerged based on fluorescence polarization, fluorescence energy transfer (TaqManTM,molecular beacons) and channelling principles. Microanalyticaldevices (microchips), high-throughput simultaneous test arrays(microarrays, gene chips), capillary electrophoretic analysisand dipstick devices have presented new challenges and requirementsfor nucleic acid detection, and fluorescent methods currentlydominate in many of these applications.

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