Use with nucleic acids

We have developed a simple method of nonisotopically labeling sample nucleic acids, which are then hybridized simultaneously to an array of unlabeled, immobilized probes. This reversed hybridization procedure thus provides identification results after a single hybridization reaction. 

In this first study, the arrays were constructed by hand spotting of genomic DNA from various bacterial species onto nitrocellulose (NC) membranes. The DNA was denatured with sodium hydroxide and fixed to the NC membranes by baking rmder vacuum at 80°C. However, the attachment of short oligonucleotides onto NC was not practical. 

For longer double-stranded nucleic acids such as cDNA [ 200 bp (base pairs) to 1500 bp], the positively charged nylon membrane easily sequestered 

Much of fhe early work relied upon hand spotting or manual application of probes using vacuum filtration devices such as the DotBlot apparatus (BioRad Laboratories) that allowed the formation of more xmiform spotting of probes in fhe form of small dofs or rectangular slots. The use of membranes for prinfed DNA arrays (often referred to as grid arrays) was subsequently developed. 

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A variety of buffers is used in electrophoresis. The selected buffer must contain ions to carry the current. Other than current-carrying capacity, the most critical criterion for buffer selection is the stability of the sample to be analyzed. Many proteins are unstable in acidic pHs, so alkaline buffers are frequently employed. Tris-(hydroxymethyl)amino methane (TRIS or THAM), sodium acetate, and ethylenedi-aminetetraacetate (EDTA) are common solutes in buffers, with pHs between 7.9 and 8.9 typical. (Refer to Chapter 5 for a discussion of buffers.) These buffers also work well with nucleic acid fragments. In addition, phosphate buffers, e.g., 10 mMK3P04, are often used with nucleic acid fragments (1.0 mM = 0.0010 M). 

Our concern in this section, however, is not the application to biopolymers of methods that are equally applicable to smaller molecules. Rather, we discuss here a totally different approach to the determination of precise three-dimensional structures of these molecules, in which NMR data play a key role. We illustrate the concept with proteins, which have yielded particularly useful information, but the general approach can also be used with nucleic acids and with complexes of a protein and a nucleic acid. 

Figure 2-15. Western blotting with non-radioactive detection. Proteins bound to a nitrocellulose membrane can be detected immunologically by methods similar to those used with nucleic acids (Figure 2-14). Antibodies against the...

Alcohol is by far the most important predpitation agent used with nucleic acids. Predpitation is usually carried out with 2-3 (v/v) of ethanol or 1 (v/v) of isopropanol in the presence of 0.1-0.5 M Na or K acetate at pH 5.0 and 0 °C salt concentrations higher than 1 M interfere with precipitation. Monovalent cations and ethanol produce a conformational change in the nudeic add which leads to precipitation. For quantitative predpitation, the mixture should be kept for 15 min at -70 °C or 30 min at -20 °C. This is particularly important for dilute solutions (< 10 pg ml-1) more concentrated solutions, above about 0.25 mg ml-1, are predpitated quantitatively even at room temperature. Sodium and potassium acetate salts in the mixture are partially predpitated with the nuddc acid, but they can be removed by washing... 

Fairly wide use has been made of preparative gel electrophoresis in protein chemistry, and in principle there is no reason why the same procedures should not be adopted for use with nucleic acids which have the advantage that much may be accomplished with very small quantities of purified material. Thus, it is relatively easy in many situations to introduce radioactive label at very high levels and specific activity, and the use of for this purpose offers a degree of sensitivity that cannot be matched in work on proteins. The extinction coefficients of nucleic acids are also very high in the ultraviolet, so that with say 20 pg in 1 ml or less it is possible to measure optical properties, thermal melting profiles, sedimentation coefficients, and even molecular weights by sedimentation equilibrium in an instrument equipped with scanner optics. Consequently, the sacrifice of resolution that, by a malign law of nature, always accompanies any attempt to scale up an analytical fractionation method is often at least partly avoided. 

Many of the techniques used in protein purification procedures have also been adapted for use with nucleic acids. For example, several types of chromatography (e.g., ion-exchange, gel filtration, and affinity) have been used in several stages of nucleic acid purification and in the isolation of individual nucleic acid sequences. Because of its speed, HPLC has replaced many slower chromatographic separation techniques when small samples are involved. 

Darden T A, L Perera, L Li and L Pedersen 1999. New Tricks for Modelers from the Crystallography Toolkit The Particle Mesh Ewald Algorithm and Its Use in Nucleic Acid Simulations. Structure with Folding and Design 7 R55-R60. 

PAMAM dendrimers have the following characteristics which are important for their use as transfection reagents. They bind and form complexes with nucleic acids, allow transfer of the DNA-dendrimer complex into the cytoplasm of the... 

Acridine dyes used as antiseptics, i.e. proflavine and acriflavine, will react specifically with nucleic acids, by fitting into the double helical structure of this unique molecule. In so doing they interfere with its function and can thereby cause cell death. 

The permeability coefficient of 2.6x 10 locm/s at 296 K measured by Deamer is sufficient to supply the enzyme in the liposomes with ADP. How could it be shown that RNA formation actually does take place in the vesicles The increase in the RNA synthesis was detected by observing the fluorescence inside the vesicles. In the interior of the liposomes, the reaction rate is only about 20% of that found for the free enzyme, which shows that the liposome envelope does limit the efficiency of the process. The fluorescence measurements were carried out with the help of ethidium bromide, a fluorescence dye often used in nucleic acid chemistry. 

Succinylated derivatives of nucleic acids may be prepared by reaction of the anhydride with available —OH groups. The reaction forms relatively stable ester derivatives that create car-boxylates on the nucleotide for further conjugation or modification (Figure 1.83). This method has been used in nucleic acid synthesis (Matteucci and Caruthers, 1980) and to derivatize nucleotide analogs such as AZT (Tadayoni et al., 1993). 

Radioactive isotopes provide a very convenient way of monitoring the fate or metabolism of compounds that contain the isotopes. When used in this way, the isotope is described as a tracer and compounds into which the radioactive atom has been introduced are said to be labelled or tagged. The labelled molecules need only comprise a very small proportion of the total amount of the unlabelled radioactive substance because they act in the same way as the non-radioactive substance but can be detected very much more easily. The varied applications of tracers in biochemistry range from studies of metabolism in whole animals or isolated organs to sensitive quantitative analytical techniques, such as radioimmunoassay. Phosphorus-32 is used in work with nucleic acids, particularly in DNA sequencing and hybridization techniques. In these instances the isotope is used as a means of visualizing DNA separations by autoradiographic techniques. 

Buta-1,3-diene (10.101, Fig. 10.24) is a gaseous chemical used heavily in the rubber and plastics industry, the presence of which in the atmosphere is also a concern. Butadiene is suspected of increasing the risks of hematopoietic cancers, and it is classified as a probable human carcinogen. Butadiene must undergo metabolic activation to become toxic the metabolites butadiene monoepoxide (10.102, a chiral compound) and diepoxybutane (10.103, which exists in two enantiomeric and one meso-form) react with nucleic acids and glutathione [160 - 163], as does a further metabolite, 3,4-epoxybutane-l,2-diol (10.105). Interestingly, butadiene monoepoxide is at least tenfold more reactive than diepoxybutane toward nucleic acids or H20. Conjugation between the C=C bond and the oxirane may account for this enhanced reactivity. 

Cellular autoradiography techniques using radioactive nucleic acid probes have several features in common with nucleic acid immunocytochemistry. The method is based on the hybridization of radioactive probes to cellular targets and the subsequent exposure of photographic emulsion, which, when developed, reveals blackened (exposed) silver grains close to the site of hybridiza-hon. Hence, cellular autoradiography techniques permit excellent specihcity and localizahon of the hybridized probe—to 1 qm when tritium is the label used in the autoradiography-based method (9). 

The mechanism of inhibition of these protozoal infections by the most active drugs, puromycin and the aminonucleoside, is not known. Puromycin and nucleocidin both interfere with protein synthesis, but the aminonucleoside does not. It is known to be demethylated to 3 -amino-3-deoxyadenosine, which is phosphorylated and interferes with nucleic acid metabolism (see above). Whether puromycin must be converted to the aminonucleoside before it can inhibit protozoa has not been established. Some purine analogues known to interfere with nucleic acid metabolism, however, are less effective as antiprotozoal agents, even in vitro, perhaps because their effects are primarily on the de novo pathway which many, if not all, protozoa do not use [406]. 

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