Nucleic acids fluorescence methods

A typical DNA array fabrication and application process involves three major steps. First, nucleic acids (the capture sequences or probes) are immobilized at discrete positions on surface activated substrates. Secondly, the resulting array is hybridized with a complex mixture of fluorescently labelled nucleic acids (the target), and thirdly subsequent to hybridization, the fluorescent markers are detected using a high-resolution scanning laser that quantifies the interaction. This chapter focuses on the first of these processes and provides the reader with an overview of substrates, surface activation methods and dehvery systems available for nucleic acid immobilization. 

Sela, L, Fluorescence of nucleic acids with ethidium bromide an indication of the configurative state of nucleic acids, Biochim. Biophys. Acta 190, 216-219, 1969 Le Pecq, J.B., Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. Methods Biochem. Anal. 20, 41-86, 1971 Borst, P., Ethidium DNA agarose gel electrophoresis how it started, lUBMB Life 57, 145-141, 2005. 

Proudnikov D, Mirzabekov A. Chemical methods of DNA and RNA fluorescent labeling. Nucleic Acids Res. 1996 24 4535-4542. 

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

Fluorescence Methods for the Analysis of Nucleic Acids in Recombinant Biological Products... 

Regulatory agencies currently set stringent standards on the quantities of nucleic acids allowed in recombinant biological products. In the pharmaceutical industry these requirements necessitate the quantification of trace amounts of nucleic acids in the presence of large quantities of protein and other excipients. Flourescence methods offer advantages for such analyses, but also have limitations. The use of a variety of fluorescent dyes and techniques is described here, and practical examples of such use are presented. 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

In current practice the fluorescence assay is often followed by the use of hybridization techniques when more selectivity is required. We have for instance used the fluorescence techniques to obtain data on the nucleic acid content of malaria vaccine proteins produced in Escherichia coli. The rapid turnaround time of the fluorescence assay is particularly useful during the early stages of purification to determine the optimal process conditions. After the final process has been arrived at and a variety of methods used to assess the nucleic acid content (including the hybridization techniques), the fluorescence method can be developed for routine quality-control purposes. In certain cases, particularly at high protein concentrations, the dye may bind to the protein with... 

In order to further extend the utility of fluorescence methods the use of time-resolution methods, fluorescence polarization, and laser techniques should be explored. The addition of other dyes with enhanced fluorescence properties on binding and increased selectivity to various types of nucleic acids will be necessary to further develop more useful analytical methods. 

Clegg, R. M. (1992). Fluorescence resonance energy transfer and nucleic acids. Methods. Enzymol. 211, 353-358. 

Ha, T. (2001). Single-molecule fluorescence methods for the study of nucleic acids. Curr. Opin. Struct. Biol. 11, 287-92. 

Recently, the use of AR has extended into several other areas, yielding interesting information for cytology, fresh cell/tissue sections, and fluorescence IHC (fluorescence in situ hybridization [FISH]), in addition to adaptations of the method for extraction of nucleic acids and proteins from FFPE tissues for use with modern methods of molecular analysis. In this chapter, the emphasis is on expanded applications in diagnostic cytology, fresh frozen cell/... 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Urdea, M.S., Warner, B.D., Running, J.A., Stempien, M., Clyne, J., and Horn, T. (1988) A comparison of non-radioactive hybridization assay methods using fluorescent, chemiluminescent and enzyme-labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 16, 4937-4956. 

Fluorescence is also a powerful tool for investigating the structure and dynamics of matter or living systems at a molecular or supramolecular level. Polymers, solutions of surfactants, solid surfaces, biological membranes, proteins, nucleic acids and living cells are well-known examples of systems in which estimates of local parameters such as polarity, fluidity, order, molecular mobility and electrical potential is possible by means of fluorescent molecules playing the role of probes. The latter can be intrinsic or introduced on purpose. The high sensitivity of fluo-rimetric methods in conjunction with the specificity of the response of probes to their microenvironment contribute towards the success of this approach. Another factor is the ability of probes to provide information on dynamics of fast phenomena and/or the structural parameters of the system under study. 

The broad field of nucleic acid structure and dynamics has undergone remarkable development during the past decade. Especially in regard to dynamics, modem fluorescence methods have yielded some of the most important advances. This chapter concerns primarily the application of time-resolved fluorescence techniques to study the dynamics of nucleic acid/dye complexes, and the inferences regarding rotational mobilities, deformation potentials, and alternate structures of nucleic acids that follow from such experiments. Emphasis is mainly on the use of time-resolved fluorescence polarization anisotropy (FPA), although results obtained using other techniques are also noted. This chapter is devoted mainly to free DNAs and tRNAs, but DNAs in nucleosomes, chromatin, viruses, and sperm are also briefly discussed. 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

The obvious similarity between the purine bases of DNA and pteridines, especially between guanosine and pterins, has encouraged extensive studies of the synthesis and properties of pteridine-containing nucleoside and nucleotides. Synthetic methods have naturally built upon established methods of nucleic acid synthesis. The primary property of use in applications of these compounds to DNA chemistry is fluorescence, which is very much greater for pteridines than for purines. 

Phosphorimetric methods have been used to determine such substances as nucleic acids, amino acids, and enzymes. However, this is not a widely used method since it cannot be run at room temperature. Measurements are usually performed with liquid nitrogen to prevent degradation due to collision deactivation. Fluorometric methods are used to determine both inorganic and organic species. Instruments used for measuring fluorescence and phosphorescence are fluorometers and spectrofluorometers, respectively. These instruments are similar to ultraviolet and visible spectrometers,... 

Currently, there is a need for high-throughput determination of nucleic acid sequences. At present, detection systems most commonly employ fluorescence-based methods. However, wide spread applications of such methods are limited by low speed, high cost, size, and number of incubations steps, among other factors. Application of electrochemical methods in affinity DNA sensors presents likely a promising alternative, allowing miniaturization and cost reduction, and potentially allowing application in point-of-care assays. 

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