Nucleic acid-based methods

Serological techniques can detect target bacteria rapidly in mixtures, but their accuracy depends on the specificity of the antibody used. The use of monoclonal instead of polyclonal antibodies may increase specificity.49,52,58 However, because the same epitope can be present in more than one species, a monoclonal antibody against one species may cross-react with other bacteria.50 For this reason serological methods are not always successful for detection of bacteria in environmental samples and nucleic acid-based methods are now commonly used. 

Two principal types of nucleic acid-based methods, nucleic acid hybridization and polymerase chain reaction (PCR), are commonly used for the rapid identification of bacteria. A few other nucleic acid-based methods will also be mentioned. 

Mothershed EA, Whitney AM. Nucleic acid-based methods for the detection of bacterial pathogens Present and future considerations for the clinical laboratory. Clin Chim Acta. 2006 363 206-220. 

Sidransky, D. 1997. Nucleic Acid-Based Methods for the Detection of Cancer. Science, November 7 1054-1058. 

Nucleic-acid based methods A number of detection and identification methods have been developed that are based on specific nucleotide sequences in RNA or DNA and several are noted in Table 2. Synopses of other related methods are presented in Table 3. 

Nucleic Acid—Based Methods Specific DNA probes have been developed to detect segments of genetic material coding for known enzymes. A gene probe developed to detect the hydrogenase enzyme which occurs broadly in SRB from the genus Desulfovibrio has been tested on samples from an oilfield waterflood plagued with iron-sulfide related corrosion problems. The enzyme was detected with this probe... 

The refinement of other analytical methods, such as electrophoresis [34,36], the various techniques of optical spectroscopy [103-105], and nuclear magnetic resonance [201], is supplemented by the recent advances in real-time affinity measurements [152,202], contributing to the understanding of biomolecular reactivity. Taken together, the improvement of analytical methods will eventually allow a comprehensive characterization of the structure, topology, and properties of the nucleic acid-based supramolecular components under consideration for distinctive applications in nanobiotechnology. 

All the nucleic acid bases absorb UV radiation, as seen in Tables 11-1, 11-2, 11-3, 11-4, and 11-5, making them vulnerable to the UV radiation of sunlight, since the energy of the photons absorbed could lead to photochemical reactions. As already mentioned above, the excited state lifetimes of the natural nucleobases, and their nucleotides, and nucleosides are very short, indicating that ultrafast radiationless decay to the ground state takes place [6], The mechanism for nonradiative decay in all the nucleobases has been investigated with quantum mechanical methods. Below we summarize these studies for each base and make an effort to find common mechanisms if they exist. 

Our method has evolved during many studies over the last two decades. These include studies on the effect of strong internal electric fields in crystals on optical transition dipole directions of nucleic acid bases [2, 3], QM-MM predictions of time-dependent solvatochromism on 3-methylindole (3MI) in water [4], and on tryptophan in several proteins [5-8]. More recently, the same techniques have been... 

To modify the unique chemical groups on nucleic acids, novel methods have been developed that allow derivatization through discrete sites on the available bases, sugars, or phosphate groups (see Chapter 1, Section 3 for a discussion of RNA and DNA structure). These chemical methods can be used to add a functional group or a label to an individual nucleotide or to one or more sites in oligonucleotide probes or full-sized DNA or RNA polymers. 

Methods currently available for chemiluminescent detection of nucleic acids are not based on derivatization techniques that directly recognize one of the nucleic acid bases or nucleotides. For chemical derivatization-based chemiluminescent detection, the specific reactivity of alkyl glyoxals and arylglyoxals with adenine or guanine nucleotides has been investigated. 

W3. Wolcott, M. J., Advances in nucleic acid-based detection methods. Clin. Microbiol. Rev. 5, 370-386 (1992). 

Carboxyethyl derivatives of the nucleic acid bases were grafted onto poly-L-lysine by using the activated ester method ( ). 

A series of new amino acid derivatives having pendant nucleic acid bases was prepared by the reaction of L-lysine and L-glutamic acid with the nucleic acid bases. These amino acids were further polymerized by using the N-carboxyamino acid anhydride ( NCA ) method. Alternatively, the nucleic acid base substituted poly-L-lysines were also prepared by using polymer reactions which include the reaction of carboxyethyl derivatives of the bases onto poly-L-lysine. Physico-chemical properties of the polymers obtained were given. 

Fenske DB, Cullis PR. Entrapment of small molecules and nucleic acid-based drugs in liposomes. Methods Enzymol 2005 391 7. 

Both approaches are simple and allow efficient encapsulation of nucleic acid-based molecules such as oligonucleotides (9,10) and pDNA (8,10,12) in liposomes that are small in size (about lOOnm diameter) and stable in circulation, protecting the cargo from degradation. In the sections to follow, we will provide a brief overview of these methods. 

The educational background of bench microbiologists, supervisors, and managers is now even more important, given the current transition from classieal to nucleic acid-based testing methods. 

Schiff base (reductive amination) method Nucleic acids Carbodiimide method... 

Another method is to produce EPR basis spectra by irradiating various nucleic acid bases. The EPR spectrum of DNA is simulated by taking various combinations of the... 

The intercalation of polycyclic aromatic compounds into duplex DNA structures was used to develop nucleic acid-based electrochemical sensors.66 For example, the bis-ferrocene-tethered naphthalene diimide (16) was used as a redox-active intercalator to probe DNA hybridization.67 The thiolated probe was assembled on a Au electrode, and the formation of the duplex DNA with the complementary analyte nucleic acid was probed by the intercalation of (16) into the double-stranded nucleic acid structure and by following the voltammetric response of the ferrocene units (Fig. 12.17a). The method enabled the analysis of the target DNA with a sensitivity that corresponded to ca. 1 x 10-20mol. 

The similar accuracies of different well-parameterized continuum models implies that they will also perform similarly for the computation of partition coefficients, and that has proven to be the case in most studies to date (see, for example, Bordner, Cavasotto, and Abagyan 2002 and Curutchet et al. 2003b). In Table 11.4 the previously presented SMx results for the chloroform/water partitioning of die methylated canonical nucleic acid bases are compared to results from die MST-ST/HF/6-31G method, and also to purely electrostatic results obtained using a multipole expansion SCRF method. As the latter does not include any accounting for non-electrostatic effects, its performance is significantly degraded compared to the other two. 

Thg IEHT method with a different set of parameters has been applied by Rein et al.212 to calculate the total charge distribution in cytosine and other nucleic acid bases (cf. Fig. 4). The 77-charges have not been indicated. 

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