Stains, labels and detection strategies for nucleic acids assays

Abstract

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 nal 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 uorophore (e.g. cyanine dyes) and enzyme class of labels (e.g. re y 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 uorescence polarization, uorescence energy transfer (TaqMan TM , molecular beacons) and channelling principles. Microanalytical devices (microchips), highthroughput simultaneous test arrays (microarrays, gene chips), capillary electrophoretic analysis and dipstick devices have presented new challenges and requirements for nucleic acid detection, and uorescent methods currently dominate in many of these applications.

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

“…Staining of nucleic acids is usually achieved by the use of fluorescent or strongly absorbing chromophores. [1] Asymmetric cyanine dyes, such as oxazole yellow (YO and its dimer YOYO), thiazole orange (TO and its dimer TOTO), SYBR Green or PicoGreen are particularly interesting because of their extraordinary increase in fluorescence upon binding to nucleic acids. [2][3][4][5][6] This property has led to advancements in countless applications in molecular biology, such as DNA quantification in the homogenous phase and in gels, real-time polymerase chain reaction, and in DNA-binding and DNAdamage assays.…”

Intracellular chemiluminescence activates targeted photodynamic destruction of leukaemic cells

“…This process is extensively employed in chemiluminescent detection techniques (Kricka, 2002;Templin et al, 2002) and in cell physiology studies (Kubo et al, 1987;Nemeth et al, 1999;Kawagae and Nakagawa, 2000) but there is no literature on the use of luminol as an energy source in the field of PDT of cancer cells. The emission spectrum of luminol has been reported to comprise two major peaks, at 424 and 485 nm (White et al, 1964).…”

“…Carpenter et al (1994) employed intracellular bioluminescent activation of hypericin and the subsequent destruction of equine dermal cells, whereas Phillip and Maximuke (1989) used a haematoporphyrin derivative (Photofrin II) and a multicomponent solution to induce intracellular CL in mammary adenocarcinomas. In searching for a simple means to induce intracellular CL, we turned to luminol, which has been successfully used in a variety of CL-based assays systems (Kricka, 2002). The mechanism of the CL reaction of luminol has been known for some time (White et al, 1964) and whereas some physico-chemical aspects of luminol activation in macrophages have been examined (Nemeth et al, 1999), the luminol -PDT connection has never been exploited to induce PDT cytotoxicity in tumour cells.…”

Adsorption-Elution Techniques and Molecular Detection of Enteric Viruses from Water

“…The rt PCR measures the amount of amplicon produced at each cycle of amplification using fluorescence based technology [34,37]. The amplicon is measured in 'real time' or as it is being produced by labelling and detecting the accumulating product with a fluorescently tagged substrate during amplification procedure [38]. In rt RT-PCR/PCR the amplicon is measured early in the reaction during the exponential phase of PCR when amplification is proceeding most efficiently [38].…”

“…The amplicon is measured in 'real time' or as it is being produced by labelling and detecting the accumulating product with a fluorescently tagged substrate during amplification procedure [38]. In rt RT-PCR/PCR the amplicon is measured early in the reaction during the exponential phase of PCR when amplification is proceeding most efficiently [38]. Therefore rt PCR has increased speed due to reduced cycle number, is more sensitive due to higher sensitivity of fluorescent dyes used for the detection of the amplicon and more specific by use of DNA/ RNA strand specific probes like Taqman or molecular beacons [33,35,36].…”