Quantitative fluorescence of DNA-intercalated ethidium bromide on agarose gels

Techniques for analyzing DNA distributions on agarose gels are examined by both two-dimensional and one-dimensional methods. It is demonstrated that very large errors in DNA concentration occur in such analyses unless (i) the electrophoresis is performed in a careful, reproducible manner, (ii) the films are calibrated with an internal standard, (iii) high resolution densitometry is used for analyzing the films, and (iv) appropriate background controls are used to determine the baselines for integration. Two-dimensional scanning produces more accurate results than one-dimensional scanning, but in cases where the bands are relatively uniform, the one-dimensional analysis gives good results. A technique for determining accurate distributions is described.     

Covalent attachment of synthetic DNA to self-assembled monolayer films.

The covalent attachment of thiol-modified DNA oligomers; to self-assembled monolayer silane films on fused silica and oxidized silicon substrates is described. A heterobifunctional crosslinking molecule bearing both thiol- and amino-reactive moieties was used to tether a DNA oligomer (modified at its terminus with a thiol group) to an aminosilane film formed on silica surfaces. A variety of aminosilanes, crosslinkers and treatment conditions have been tested to identify optimal conditions for DNA immobilization using this approach. The DNA films which result have been characterized using UV spectroscopy, water contact angle measurement, radiolabeling and hybridization methods.     

Modulation of the B-A transition of DNA by potential antitumor antibiotics. Influence of the base composition of DNA

The B-A transition of DNA in oriented films of DNA-drug complexes is more or less restricted as a consequence of drug binding as revealed by infrared linear dichroism. A fraction of DNA is irreversibly locked into the B form. This behavior is described by the number of DNA base pairs "frozen" in the B form by one drug molecule. This quantity is dependent on the DNA sequence the drug is attached to. In this paper, drug complexes of oriented films of NaDNA with a GC content of 42% from calf thymus and a GC-rich DNA from Micrococcus lysodeikticus were compared. The restriction of the B-A transition of DNA complexes with two intercalating antibiotics, aclacinomycin A and violamycin BI, is not severely influenced by the base composition of DNA. By contrast, the strong groove binding oligopeptide antibiotics netropsin and distamycin A are much less effective to restrict the B-A transition of GC-rich DNA than of AT-rich DNA. This finding is in agreement with previous results by other methods which support a model based upon a strong preference of AT clusters by these two non-intercalating drugs.     

Construction of linear functional expression elements with DNA and RNA hybrid primers: a flexible and fast method for proteomics

Two methods of constructing linear functional expression elements (LFEE) using hybrid DNA and RNA primers in DNA amplification for rapid gene expression are described. In both methods, it is not necessary to have additional transformation or bacterial propagation. The promoter, open reading frame (ORF) and terminator are amplified using Pfu or Taq DNA polymerase. Three elements containing DNA or RNA overhang are covalently ligated by T4 DNA ligase. The recombinant molecule is amplified with element-specific primers. The LFEE can be generated by both methods in a few hours and can be expressed in mammalian cells.     

A rapid method for high throughput DNA extraction from plant material for PCR amplification

A rapid and reliable method is described for high throughput extraction of DNA from plant material using glass beads in a flat-bottomed microtitre plate. This procedure is quick, inexpensive, and allows up to 96 samples to be processed in parallel. PCR products produced by the recovered DNA are consistently equivalent to those produced through traditional extraction methods.     

A novel method for preparing single-stranded DNA for pyrosequencing

Pyrosequencing technology is a powerful genotyping tool that requires the generation of single stranded DNA. Currently, two simple, solid-phase-based methods are available for this, but they require special equipment, they are not automated, and they are relatively expensive because of the need for biotinylated polymerase chain reaction primers. In this article, an enzymatic liquid-phase method for the generation of high-quality, single-stranded DNA, and its novel use for Pyrosequencing are described. The method has also been fully automated.     

Species differentiation by DNA-modified carbon electrodes using an ac impedimetric approach

A simple and novel electrochemical biosensor based approach is described for differentiating between differing species of fish on the basis of DNA hybridisation events. Screen-printed carbon electrodes modified with a variety of polymers were used to immobilise commercially available DNA in a single-stranded form. AC impedimetric measurements were firstly carried out on these systems and then upon exposure to single-stranded DNA solutions. When the electrode and solution DNA were complementary, a large drop in impedance was measured; this did not occur for non-matching DNA exposures. DNA hybridisation sensors for closely related species of fish were in the first instance developed as a demonstration for this approach. Species of fish such as herrings and salmon could be differentiated by this method. This sensor format offers great promise for many DNA hybridisation applications and lends itself to mass fabrication due to the simplicity and inexpensiveness of the materials and methods used. The hybridisation results were confirmed by use of ellipsometry to measure the characteristics of similar films deposited on silicon substrates.     

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

DNA isolation methods for medicinal and aromatic plants

Several protocols described for plant DNA isolation fail to produce good quality DNA from medicinal herbs and aromatic plants. These plants contain exceptionally high amounts of secondary metabolites that interfere with DNA isolation. To address this problem, we developed 2 DNA isolation methods for sundew and tarragon that produce DNA suitable for molecular biological applications. One of the methods also is applicable for milfoil and Siberian ginseng.     

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.     

Methods for selection of aptamers to protein targets

Aptamers are synthetic single-stranded RNA or DNA molecules capable of specific binding to other target molecules. In this review, the main aptamer properties are considered and methods for selection of aptamers against various protein targets are described. Special attention is given to the methods for directed selection of aptamers, which allow one to obtain ligands with specified properties.     

用于PCR扩增的DNA简易制备法现状

介绍了 6种用于PCR扩增的DNA简易制备法。所得DNA质量符合PCR扩增之用 ,经过试验也可用于系统生物学之研究     

Development and application of a nonradioactive phosphorescent autoradiograph marker

A simple and rapid method for permanently marking autoradiographs is described. This procedure is based on the phosphorescence of light-activated zinc sulfide and the subsequent exposure of x-ray films by this light emission. A lacquer-based carrier allows the zinc sulfide to remain in suspension and permits permanent marking onto diverse laboratory substrates such as x-ray films, paper, plastic wraps and nitrocellulose- and nylon-based membranes. An analogous wax-based carrier allows marking onto paper and dried acrylamide gels, which is useful for processing large numbers of radioactive DNA sequencing gels on a high-throughput scale. These inexpensive and nonhazardous markers will be useful in protocols that use x-ray films, regardless of whether radioactive or nonradioactive detection systems are used.     

Simplified method for isolation of white cells from whole blood suitable for direct polymerase chain reaction

A simple method for isolating mononuclear cells from whole blood is described. The procedure utilizes phytohemagglutinin to agglutinate the erythrocytes, separating white cells from whole blood in a very brief handling time. The isolated cells are readily subjected to DNA isolation simply by boiling, and the released DNA can be directly employed for the polymerase chain reaction analysis. The efficiency of this method is similar to other conventional methods, but less costly and less time-consuming. This method is particularly useful in analyzing DNA samples from the peripheral blood cells when the simplicity and low cost of the assay are preferable.     

Structured thin films as functional components within biosensors

This review provides an introduction to the field of thin films formed by Langmuir-Blodgett or self-assembly techniques and discusses applications in the field of biosensors. The review commences with an overview of thin films and methods of construction. Methods covered will include Langmuir-Blodgett film formation, formation of self-assembled monolayers such as gold-thiol monolayers and the formation of multilayers by the self-assembly of polyelectrolytes. The structure and forces governing the formation of the materials will also be discussed. The next section focussed on methods for interrogating these films to determine their selectivity and activity. Interrogation methods to be covered will include electrochemical measurements, optical measurements, quartz crystal microbalance, surface plasmon resonance and other techniques. The final section is dedicated to the functionality of these films, incorporation of biomolecules within these films and their effect on film structure. Species for incorporation will include antibodies, enzymes, proteins and DNA. Discussions on the location, availability, activity and stability of the included species are included. The review finishes with a short consideration of future research possibilities and applications of these films.     

Sensors for toxicity of chemicals and oxidative stress based on electrochemical catalytic DNA oxidation

Films of DNA, enzymes, polyions, and catalytic redox polyions of nanometer thickness on electrodes can provide active elements for sensors for screening the toxicity of chemicals and their metabolites, and for oxidative stress. The unifying feature of this approach involves layer-by-layer electrostatic assembly of films designed to detect DNA damage. Films containing DNA and enzymes enable detection of structural damage to DNA as a basis for toxicity screening. These films bioactivate chemicals to their metabolites, which can then react with DNA, mimicking toxicity pathways in the human liver. Metallopolyions that catalyze DNA oxidation can be incorporated into DNA/enzyme films leading to "reagentless" sensors. These sensors are suitable for detecting relative DNA damage rates in <5 min of the enzyme reactions. Films of the osmium polymer [Os(bpy)(2)(PVP)(10)Cl](+) [poly(vinylpyridine), PVP] can be used to monitor DNA oxidation selectively. Such films may be applicable to determination of oxidized DNA as a clinical biomarker for oxidative stress. Inclusion of the analogous ruthenium metallopolymer in the sensor provides a monitor for oxidation of other nucleobases.     

DNA complexes, formed on aqueous phase surfaces: new planar polymeric and composite nanostructures

The formation of DNA complexes with Langmuir monolayers of the cationic lipid octadecylamine (ODA) and the new amphiphilic polycation poly-4-vinylpyridine with 16% of cetylpyridinium groups (PVP-16) on the surface of an aqueous solution of native DNA of low ionic strength was studied. Topographic images of Langmuir-Blodgett films of DNA/ODA and DNA/PVP-16 complexes applied to micaceous substrates were investigated by the method of atomic force microscopy. It was found that films of the amphiphilic polycation have an ordered planar polycrystalline structure. The morphology of planar DNA complexes with the amphiphilic cation substantially depended on the incubation time and the phase state of the monolayer on the surface of the aqueous DNA solution. Complex structures and individual DNA molecules were observed on the surface of the amphiphilic monolayer. Along with quasi-linear individual bound DNA molecules, characteristic extended net-like structures and quasi-circular toroidal condensed conformations of planar DNA complexes were detected. Mono- and multilayer films of DNA/PVP-16 complexes were used as templates and nanoreactors for the synthesis of inorganic nanostructures via the binding of metal cations from the solution and subsequent generation of the inorganic phase. As a result, ultrathin polymeric composite films with integrated DNA building blocks and quasi-linear arrays of inorganic semiconductor (CdS) and iron oxide nanoparticles and nanowires were obtained. The nanostructures obtained were characterized by scanning probe microscopy and transmission electron microscopy techniques. The methods developed are promising for investigating the mechanisms of structural organization and transformation in DNA and polyelectrolyte complexes at the gas-liquid interface and for the design of new extremely thin highly ordered planar polymeric and composite materials, films, and coatings with controlled ultrastructure for applications in nanoelectronics and nanobiotechnology.     

A method for sequencing polymerase chain reaction products can be used to sequence Bacillus subtilis "miniprep" plasmid DNA

Several methods for sequencing double-stranded plasmid DNA isolated from E. coli have been described. These methods are usually not effective when used to sequence plasmid DNA isolated from Bacillus subtilis. In the course of developing a simplified version of a previously published protocol for polymerase chain reaction product sequencing, it was found that this protocol could be used for sequencing plasmid DNA isolated from Bacillus subtilis.     

Stochastic models for heterogeneous DNA sequences

The composition of naturally occurring DNA sequences is often strikingly heterogeneous. In this paper, the DNA sequence is viewed as a stochastic process with local compositional properties determined by the states of a hidden Markov chain. The model used is a discrete-state, discreteoutcome version of a general model for non-stationary time series proposed by Kitagawa (1987). A smoothing algorithm is described which can be used to reconstruct the hidden process and produce graphic displays of the compositional structure of a sequence. The problem of parameter estimation is approached using likelihood methods and an EM algorithm for approximating the maximum likelihood estimate is derived. The methods are applied to sequences from yeast mitochondrial DNA, human and mouse mitochondrial DNAs, a human X chromosomal fragment and the complete genome of bacteriophage lambda.     

A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes.

A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.