Fluorescent Labeling of DNA

The ligation methodology has been successfully applied to attaching various moieties to cell surfaces fluorescent labeling of DNA fragments... 

Fig. 8 CuAAC reaction for in vivo labeling of DNA and RNA. a Schematic representation for in vivo fluorescent labeling of DNA and RNA molecules using metaboUcally incorporated EdU (or EU) and CuAAC reaction, b Detection of vims infection using the ceU-selective incorporation of dF-EdU and CuAAC reaction [102-104]. CuAAC copper(I)-catalyzed azide-aUcyne cycloaddition, EdU 5-ethynyl-2-deoxyuridine, EU 5-ethynyluridine, dF-EdU 2 -deoxy-2, 2 -difluoro-5-ethynyluridine...

Nucleic acid (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) probes utilize labeled, ie, radioactive, enzymatic, or fluorescent, fragments of DNA or RNA (the probe) to detect complimentary DNA or RNA sequences in a sample. Because the probe is tailored for one specific nucleic acid, these assays are highly specific and very sensitive (45). 

Rhodamine B 67 is frequently used in the quantitative determination of DNA or RNA and fluorescent labeling for DNA [190-192]. This dye was assembled onto the surface of a quartz substrate by electrostatic interaction between the fluorescence reagent RB and y-aminopropyltriethoxysilane (APES), and the Quartz/ APES/RB film was constructed (Fig. 1) [193]. 

Fluorescent labelling of protein/RNA or DNA combined with optical or confocal microscopy imaging is a powerful widely used imaging technique that can (like Raman spectroscopy) also be applied to live cells or tissues. This technique, however, is limited by the number of fluorescent molecular... 

However, real-time detection requires access to a special real-time PCR cycler, which is able to detect the increase/decrease of added fluorescence labels during DNA amplification. Although these machines are more and more common for quantitative DNA analysis, their availability in clinical laboratories is still limited. Therefore, the following subsections also include a detailed overview of the classical approaches to quantitative (I)PCR amplificate, analysis which exchanges less demanding PCR equipment for additional hands-on time. The sensitivity of real-time or end-point IPCR detection is quite similar. A comparison of the influence of different endpoint detection methods to the overall sensitivity of IPCR is given in Fig. 5. 

Pljevaljcic G, Pignot M, Weinhold E. Design of a new fluorescent cofactor for DNA methyl transferase and sequence-specific labeling of DNA. J. Am. Chem. Soc. 2003 125 3486-3492. 

To show that dehybridization occurs on a reasonable timescale in these membranes, we exposed a hairpin-DNA membrane to a fluorescently labeled version of the PC-DNA (Table 24.1). The membrane was then rinsed with buffer solution and immersed into a solution of either pure buffer or buffer containing unlabeled PC-DNA. If the dehybridization reaction is facile, the fluorescently labeled PC-DNA should be released into the solution. We found that dehybridization does occur, but it is strongly accelerated when unlabeled PC-DNA is present in the solution (Figure 24.9). Hence dehybridization is much faster when it occurs by a cooperative process whereby one PC-DNA molecule displaces another from an extant duplex [46]. 

FIGURE 24.9 Release of fluorescently labeled PC-DNA from a membrane containing the hairpin-DNA transporter. The fluorescently labeled PC-DNA was released into a buffer solution containing no unlabeled PC-DNA (lower curve) or into a buffer containing 9 p,M unlabeled PC-DNA (upper curve). (From Kohli, P., Harrell, C.C., Cao, Z., Gasparac, R., Tan, W., and Martin, C.R., Science, 305, 984, 2004. With permission.)... 

T4 RNA ligase can introduce fluorescent 3 -O-(5 -phos-phoryldeoxycytidyl) S-bimane phosphorothioate into RNA or DNA (Cosstick et al., 1984). Other methods have been developed for fluorochrome labeling of DNA for automated sequencing, either by labeled primers or dye-terminators (fluorescent ddNTP Applied Biosystems) with DNA polymerase or with terminal dNTP transferase (Trainor and Jensen, 1988). We successfully used fluorescein-dUTP (Boehringer Mannheim, soon available), both for automated sequencing and probe preparation. All common DNA polymerases incorporate this analogue efficiently. 

A set of fluorophores derived from naphthalene, phenanthrene, pyrene, phenazine and fluorene have been conjugated to the 5 -ends of DNA and RNA to compare their physico-chemical properties. Decreasing the Ji-electron density led to an enhancement in thermal stability, attributable to more favourable Jt-Ji interactions. Stability is further enhanced by using nitrated fluorophores. Fluorescent labelling of ODNs using oxyamino modified fluorescein has been reported by the incorporation into DNA of aldehyde functions. The aldehyde function was attached either at the 5 -end via a phosphate linker or internally via 8-mercaptobutanal. Reduction of the resulting oxime was not necessary. 

Usually, the detection of pathogenic bacteria, such as Escherichia coli is based on the selective growth of these bacteria in liquid media or on plates. This procedure may require several days [52]. More recently, methods such as pathogen recognition by fluorescently labeled antibodies, DNA probes, or bacteriophages have been developed and proved to be much faster [52],... 

Fluorescence labeling of nucleic acids is limited to double-stranded structures. Both double-stranded DNA and RNA react with ethidium bromide under the formation of a fluorescent product (360/580) [206-208]. The sensitivity of this method is 50 /ig for DNA and 100 jag for RNA. The differentiation between RNA and DNA can be done by using specific nucleases. According to Jovin [209] it is also possible to add ethidium bromide at a low concentration (0.1 relative to DNA phosphorus) before electrophoresis without any effect upon the subsequent separation. 

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