Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences

The existence of hypervariable DNA sequences in nuclear genomes, and the use of appropriate "fingerprinting" probes to detect them, has gained widespread scientific interest, and also led to multiple applications in diverse areas. Two years ago, the new technique of "DNA fingerprinting" was also introduced into the analysis and characterization of plant genomes, initially by using human or M13 minisatellites as probes. In the present article, we demonstrate the applicability for plant DNA fingerprinting of oligonucleotide probes specific for simple repetitive DNA sequences. We show that various levels of intra- and interspecific polymorphisms can be detected; the information to be gained depends on the optimal combination of probe and species. Variety-specific patterns were obtained in several cases. Some probes revealed variability between individuals. Somatic variability was not observed. Different DNA isolation and purification procedures were tested in order to introduce a fast and easy-to-perform isolation method suitable for a large variety of plant species. Nonradioactive fingerprinting was performed using digoxigenated oligonucleotides as probes. Banding patterns obtained with radioactive and digoxigenin-based labeling techniques proved to be of similar quality.     

Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing

This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides, (Py)U and (Py)C, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The fluorescence spectrum of the duplex containing a (Py)U/A base pair showed a strong emission at 397 nm on 327 nm excitation. In contrast, the fluorescence of duplexes containing (Py)U/N base pairs (N = C, G, or T) was considerably weaker. The proposed structure of the duplex containing a matched (Py)U/A base pair suggests that the high polarity near the pyrenecarboxamide group is responsible for the strong A-selective fluorescence emission. Moreover, the fluorescence of the duplex containing a (Py)U/A base pair was not quenched by a flanking C/G base pair. The fluorescence properties are quite different from previous BDF nucleobases, where fluorescence is quenchable by flanking C/G base pairs. The duplex containing the C derivative, (Py)C, selectively emitted fluorescence when the base opposite (Py)C was G. The drastic change of fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. (Py)U- and (Py)C-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing of DNA samples containing c-Ha-ras and BRCA2 SNP sites.     

Oligonucleotide fingerprinting of arrayed genomic DNA sequences using LNA-modified hybridization probes

Recently, we established a robust method for the detection of hybridization events using a DNA microarray deposited on a nanoporous membrane. Here, in a follow-up study, we demonstrate the performance of this approach on a larger set of LNA-modified oligoprobes and genomic DNA sequences. Twenty-six different LNA-modified 7-mer oligoprobes were hybridized to a set of 66 randomly selected human genomic DNA clones spotted on a nanoporous membrane slide. Subsequently, assay sensitivity analysis was performed using receiver operating characteristic (ROC) curves. Comparison of LNA-modified heptamers and DNA heptamers revealed that the LNA modification clearly improved sensitivity and specificity of hybridization experiment. Clustering analysis was applied in order to test practical performance of hybridization experiments with LNA-modified oligoprobes in recognizing similarity of genomic DNA sequences. Comparing the results with the theoretical sequence clusters, we conclude that the application of LNA-modified oligoprobes allows for reliable clustering of DNA sequences which reflects the underlying sequence homology. Our results show that LNA-modified oligoprobes can be used effectively to unravel sequence similarity of DNA sequences and thus, to characterize the content of unknown DNA libraries.     

Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis

A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex.Abbreviations BGE background electrolyte - CE capillary electrophoresis - Epo Erythropoietin - Fab antigen-binding fragment - FITC fluorescein isothiocyanate - IEF isoelectric focusing - mAb monoclonal antibody - PBS phosphate-buffered saline - rHuEpo recombinant human erythropoietin - scFv (recombinant) single chain variable fragment - SDS-PAGE denaturing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - ECL enzyme-coupled chemoluminescence - v_H variable domain - c_H1–3 constant domains of an antibody's heavy chain     

The relevance of the complementary spherical electron density model to organometallic intermediates involved in homogeneous catalysis

The implications of the Complementary Spherical Electron Density Model, originally developed by Mingos and Hawes in 1985 and amplified in 2004, to co-ordinatively unsaturated intermediates in homogenous catalytic processes are discussed. The geometric consequences of the model for 16 and 14 electron complexes are particularly important and are supported by numerous recent X-ray crystallographic investigations. The character of the important frontier orbitals have been explored using density functional calculations.     

Redox reactions of basic fuchsin in homogeneous aqueous media

The pulse-radiolysis technique has been employed to understand the reaction mechanism and to characterize the transient species involved in the redox processes taking place in the radiation chemistry of basic fuchsin (BF⁺). One-electron reduction and oxidation reactions of BF⁺ have been carried out in homogeneous aqueous solutions employing various reducing (e _aq ^– , (CH₃)₂COH, CO ₂ ^– ) and oxidizing (N ₃, Br, Cl ₂ ^– Br ₂ ^– ) radicals. The absorption spectra of the transients formed in the above reactions have been attributed to semi-reduced and semi-oxidized species of BF⁺, respectively. The kinetic and spectroscopic properties of these transients have been evaluated. The reaction with H and OH radicals have also been performed and compared with those of specific one-electron reducing and oxidizing radicals. These reactions have been inferred predominantly by addition to BF⁺. Protolytic equilibria involving semi-reduced species of basic fuchsin have been studied over the pH range from 2 to 10 and the pK _a has been determined to be 3.9.     

Sequence-specific single-molecule analysis of 8-oxo-7,8-dihydroguanine lesions in DNA based on unzipping kinetics of complementary probes in ion channel recordings

Translocation measurements of intact DNA strands with the ion channel α-hemolysin (α-HL) are limited to single-stranded DNA (ssDNA) experiments as the dimensions of the channel prevent double-stranded DNA (dsDNA) translocation; however, if a short oligodeoxynucleotide is used to interrogate a longer ssDNA strand, it is possible to unzip the duplex region when it is captured in the α-HL vestibule, allowing the longer strand to translocate through the α-HL channel. This unzipping process has a characteristic duration based on the stability of the duplex. Here, ion channel recordings are used to detect the presence and relative location of the oxidized damage site 8-oxo-7,8-dihydroguanine (OG) in a sequence-specific manner. OG engages in base pairing to C or A with unique stabilities relative to native base Watson-Crick pairings, and this phenomenon is used here to engineer probe sequences (10-15mers) that, when base-paired with a 65mer sequence of interest, containing either G or OG at a single site, produce characteristic unzipping times that correspond well with the duplex melting temperature (T(m)). Unzipping times also depend on the direction from which the duplex enters the vestibule if the stabilities of leading base pairs at the ends of the duplex are significantly different. It is shown here that the presence of a single DNA lesion can be distinguished from an undamaged sequence and that the relative location of the damage site can be determined based on the duration of duplex unzipping.     

Transformations of diepoxy derivatives of limonene in homogeneous acidic media

Transformations of diepoxy derivatives of limonene under conditions of homogeneous acid catalysis were studied, and their results were compared with those obtained over heterogeneous catalysts. A number of previously unknown compounds were isolated. The most possible transformation pathways were analyzed by computer simulation using molecular-mechanics and quantum-chemical methods.     

DNA binding hydroxyl radical probes

The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores, which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.     

Simple Michael acceptor type coumarin derived turn-on fluorescence probes to detect cyanide in pure water

The present article describes design and synthesis of coumarin derived chemical reaction based chemodosimeter to recognize cyanide in aqueous medium. The Michael acceptor type coumarin derived probes 3 and 4 upon interaction with cyanide undergo Michael type nucleophilic addition reaction to form adducts in which fluorescence intensity was significantly enhanced, ‘turn-on’ by the change in intramolecular charge transfer (ICT) mechanism. The nonfluorescent color of solutions changed to a naked-eye sensitive fluorescent blue (switched-on). Job’s plot analysis revealed a 1:1 stoichiometry for the interaction between the probe and cyanide anion with a high detection limit.     

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.     

Optimized oligonucleotide probes for DNA fingerprinting

The three different simple repetitive oligonucleotide probes (CT)8, (CAC)5 and (TCC)5 were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases Alu I, Hinf I and Mbo I. The resulting DNA fingerprints were analyzed and different parameters calculated, such as the maximal mean allele frequency and the average number of polymorphic bands per individual. The highest number of bands was obtained after hybridization of Hinf I digested DNA with (CAC)5. The probability of finding the same band pattern as in individual A in individual B is 2 x 10(-8). The DNAs of monozygous twins show indistinguishable banding patterns and the bands are inherited according to the Mendelian laws. Thus this procedure reveals informative fingerprints that can be used for individual identification, e.g. in paternity testing and in forensic applications. In most of these experiments 32P-labelled probes were employed, yet the biotinylated oligonucleotide (GACA)4 produced results which were equivalent to those obtained by hybridization with the 32P-labelled probe (GACA)4.     

Olefin metathesis in homogeneous aqueous media catalyzed by conventional ruthenium catalysts

Olefin metathesis in aqueous solvents is sought for applications in green chemistry and with the hydrophilic substrates of chemical biology, such as proteins and polysaccharides. Most demonstrations of metathesis in water, however, utilize exotic complexes. We have examined the performance of conventional catalysts in homogeneous water/organic mixtures, finding that the second-generation Hoveyda-Grubbs catalyst has extraordinary efficiency in aqueous dimethoxyethane and aqueous acetone. High (71-95%) conversions are achieved for ring-closing and cross metathesis of a variety of substrates in these solvent systems.     

Stereoselective homogeneous catalytic hydrogenation of disubstituted alkynes in aqueous-organic biphasic media

Summary Diphenylacetylene and 1-phenyl-1-propyne were hydrogenated to the corresponding 1,2-disubstituted alkenes in aqueous organic biphasic media using the water-soluble catalyst [{RuCl₂(mtppms)₂}₂] and an excess of the sulfonated phosphine ligand. The stereoselectivity of the reaction strongly depends on the pH of the catalyst-containing aqueous phase and under acidic conditions Z-alkenes can be obtained with a selectivity close to 100%.     

Models of slow rotational mobility of paramagnetic probes in glassy media

Experimental ESR spectra of the 2,2,6,6-tetramethyl-4-oxopiperidinoxyl (TEMPON) radical probe in the glycerol, polystyrene, and polyvinylbutyral matrices measured in the temperature range 77–373 K were quantitatively compared with the ESR spectra calculated using the known theoretical models of rotational mobility. It was shown that simulation of ESR spectra by the nonlinear least-squares method is an efficient procedure for discriminating between theoretical models. The temperature ranges were determined in which it is possible to achieve quantitative agreement between experimental and theoretical spectra as well as the ranges in which theoretical models are insufficient to quantitatively describe the experimental results. It was established that the widely used model of Brownian diffusion in isotropic medium is inadequate to describe the ESR spectra in the case of slow motions of small probe molecules. It was found that specific interactions (formation of weak complexes) between the probe molecules and the molecules of the medium results in strongly anisotropic molecular rotational motions. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1065–1073, June, 2000.     

Hybridization of DNA to bead-immobilized probes confined within a microfluidic channel

We report the factors influencing the capture of DNA by DNA-modified microbeads confined within a microfluidic channel. Quantitative correlation of target capture efficiency to probe surface concentration, solution flow rate, and target concentration are discussed. The results indicate that the microfluidic system exhibits a limit of detection of approximately 10(-10) M (approximately 10(-16) mol) DNA and a selectivity factor of approximately 8 x 10(3). Typical hybridization times are on the order of minutes.     

Branchpoint expansion in a fully complementary three-way DNA junction

Branched nucleic acid molecules serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resolution single-molecule FRET, time-resolved spectroscopy, and molecular modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in solution. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the molecule. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramolecular receptors and nanosystems.