Stains, labels and detection strategies for nucleic acids assays.
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 (TaqMan, molecular beacons) and channelling principles. Microanalytical devices (microchips), high-throughput simultaneous test arrays (microarrays, gene chips), capillary electrophoretic analysis and dipstick devices have presented new challenges and requirements for nucleic acid detection, and fluorescent methods currently dominate in many of these applications.
Kricka
Larry J
LJ
Department of Pathology, University of Pennsylvania, Philadelphia 19104, USA. kricka@mail.med.upenn.edu
D000469:Alkaline Phosphatase / Q000032:analysis; D004396:Coloring Agents / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis*; D009195:Peroxidase / Q000032:analysis; D011868:Radioisotopes / Q000032:analysis; D013194:Staining and Labeling / Q000379:methods* / Q000639:trends
D004396:Coloring Agents; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D011868:Radioisotopes; D009195:Peroxidase; D000469:Alkaline Phosphatase