DNA gel particles: An overview

A general understanding of interactions between DNA and oppositely charged compounds forms the basis for developing novel DNA-based materials, including gel particles. The association strength, which is altered by varying the chemical structure of the cationic cosolute, determines the spatial homogeneity of the gelation process, creating DNA reservoir devices and DNA matrix devices that can be designed to release either single- (ssDNA) or double-stranded (dsDNA) DNA. This review covers recent developments on the topic of DNA gel particles formed in water–water emulsion-type interfaces. The degree of DNA entrapment, particle morphology, swelling/dissolution behavior and DNA release responses are discussed as functions of the nature of the cationic agent used. On the basis of designing DNA gel particles for therapeutic purposes, recent studies on the determination of the surface hydrophobicity and the hemolytic and the cytotoxic assessments of the obtained DNA gel particles have been also reported.     

Separation of chromosomal length DNA molecules: pneumatic apparatus for rotating gels during electrophoresis

The maximum length of DNA molecules that can be separated by gel electrophoresis can be increased greatly by periodically altering the direction of the electric field with respect to the gel by an angle that exceeds 90 degrees. One method involves rotating the gel by the desired angle in alternate directions periodically during electrophoresis. We describe a modification of the rotating gel electrophoresis apparatus developed by Serwer (Electrophoresis 1987, 8, 301-304) that uses a pneumatic rotary actuator instead of a stepping motor, hence reducing the cost by about 50%. Other advantages of our design are a lower center of gravity that makes the apparatus more stable and the removal of all electrical power from beneath the fluid-filled electrophoresis chamber. We present data demonstrating the separation of chromosomal length DNA molecules from Saccharomyces cerevisiae strain 334 into 14 resolved bands in parallel lanes.     

QUANTIFICATION OF PYRIMIDINE DIMERS AND APURINIC SITES IN DNAs OF UNIFORM LENGTH

Abstract— A new simple in vitro assay for the determination of pyrimidine dimers and/or apurinic/apyrimidinic sites in non-radioactive DNA has been developed. In this procedure, DNA substrates of uniform length-which may be supercoiled, partially relaxed, relaxed or linear-are treated with agents which produce specific single strand nicks at the site of the lesion. The number of lesions per molecule can be expressed as a function of the amount of single-stranded molecules left intact after the specific nicking treatment. Unreacted molecules, which retain the original uniform length, are separated from the other smaller reaction products by electrophoresis on an alkaline agarose gel. In the case of circular molecules, the substrate is linearized by the use of an appropriate restriction endonuclease before loading on the gel. The amount of intact DNA molecules is obtained by integrating the corresponding peak of absorption after densitometric scan of the negative of the gel picture. This assay can detect up to eight damaged sites per duplex molecule. This method could be particularly useful when dealing with mixtures of DNA with different degrees of supercoiling or for comparisons between linear and circular DNA substrates.     

Nanoparticle-DNA conjugates bearing a specific number of short DNA strands by enzymatic manipulation of nanoparticle-bound DNA

Self-assembling of metallic nanoparticles to form well-defined nanostructured structures is a field that has been receiving considerable research interest in recent years. In this field, DNA is a commonly used linker molecule to direct the assembly of the nanoscale building blocks because of its unique recognition capabilities, mechanical rigidity, and physicochemical stability. This study reported our novel approach to generate gold nanoparticle-DNA conjugates bearing specially designed DNA linker molecules that can be used as building blocks to construct nanoassemblies with precisely controlled structure or as nanoprobes for quantitative DNA sequence detection analysis. In our approach, gold nanoparticle-DNA conjugates bearing a specific number of long double-stranded DNA strands were prepared by gel electrophoresis. A restriction endonuclease enzyme was then used to manipulate the length of the nanoparticle-bound DNA. This enzymatic cleavage was confirmed by gel electrophoresis, and digestion efficiency of 90% or more was achieved. With this approach, nanoparticle conjugates bearing a specific number of strands of short DNA with less than 20-base can be achieved.     

Analysis of limiting factors of DNA band separation by a DNA sequencer using fluorescence detection.

The factors affecting the electrophoretic separation of DNA bands in DNA base sequencing using fluorescence detection are analyzed. All the factors contributing to DNA band spacing and band width are evaluated; DNA diffusion and thermal effects on gels are the main considerations. The dependence of the gel's electrical resistivity on gel temperature and the variation of temperature over gel thickness are associated with a broadening of DNA band width. As a result of the analyses the maximum separable base number is represented as a function of various electrophoretic variables. The best separations are possible with an electric field strength corresponding to gel thickness. The maximum separable base number increases as the gel thickness decreases. It also increases as the migration distance increases, but it becomes saturated and has an upper limit when the migration distance is long. This upper limit increases as gel thickness decreases. DNA fragments with 600 and 601 bases can be completely separated from each other under optimum conditions for a 0.2 mm thick gel plate. Furthermore, using the band spacing information, under the same conditions, 750 bases could be assigned separately.     

Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer

In polyacrylamide gels, we have quantitatively determined Forster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein-DNA complexes. The donor-dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change. The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Forster distance R0 of 46 A compared to 50 A in solution. We have determined global structural properties of two proteins binding to double-labeled DNA using a novel gel-based fluorescence resonance energy transfer assay. In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA. The measured Forster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex.     

Maximum entropy image reconstruction of DNA sequencing gel autoradiographs

DNA sequencing gel autoradiographs become increasingly difficult to read as one moves up the gel, due to crowding and overlapping of the bands. Maximum entropy image reconstructions of the autoradiograph improve the ease with which crowded sequence data can be read, and extend the region of the gel in which reading is possible. Superior reconstructions are obtained by using nonuniform models based on the initial reconstructions.     

Monitoring of single nicks in duplex DNA by gel electrophoretic mobility-shift assay

We demonstrate that the gel electrophoretic mobility-shift assay (EMSA) can be used for site-selective and quantitative monitoring of nicks in linear double-stranded DNA (dsDNA) thus allowing to expediently follow the nicking activity of enzymes or other agents targeted to a designated dsDNA site. At elevated temperature and/or in the presence of urea, DNA fragments carrying a single nick produced by the nicking enzyme N.BstNBI exhibit a well-detectable gel retardation effect. On the basis of permutation analysis, the decreased electrophoretic mobility of nicked dsDNA fragments is attributed to a bend (or hinge) in the DNA double helix sequence-specifically generated by a nick. Since nick-induced DNA bending depends on interaction between base pairs adjacent to a nick, the change in mobility is different for nicked DNA sites with different sequences. Therefore, EMSA monitoring of differential mobility change caused by nicks within various DNA sequences could be useful for studying the differential base stacking and nearest-neighbor energetics.     

DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system

We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDA) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDA system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis.     

Movement of fluorescence pattern after photobleaching: an accelerated procedure for DNA electrophoretic mobility analysis.

A new approach which is compatible with many of the existing procedures for the analysis of DNA species in gel electrophoresis is being demonstrated. It takes advantage of fluorescence photobleaching in order to create a sharp boundary between the stained and the (partially) photobleached DNA. By arbitrarily creating a stained DNA band of narrower width, the sensitivity to detect (averaged) DNA band movements has been increased. This feature permits measurements of time-dependent electrophoretic mobility over very short time periods. The approach can be used to shorten the running time of gel electrophoresis experiment and to increase the resolution because of the sharper boundary and narrower band width. With faster running time, diffusion of both DNA and dye in the gel also becomes less serious. Movement of fluorescence pattern after photobleaching also permits measurements of localized motions when the gel pores are small in comparison with DNA sizes. Experiments demonstrating some aspects of the proposed technique, as well as the anticipated limitations, are presented and discussed.     

An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection

In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.     

2-甲基-1,4-萘醌对DNA光敏损伤的诱导作用

选择波长337 nm的激光作为激励光源,借助凝胶电泳研究了2-甲基-1,4-萘醌诱导的DNA光敏损伤.结果表明:在氧气饱和、脱氧条件下光敏损伤显著,DNA损伤主要与光子剂量、核酸与萘醌浓度比及DNA存在形式有关.     

Methods to electrophoretically stretch DNA: microcontractions, gels, and hybrid gel-microcontraction devices

The ability to controllably and continuously stretch large DNA molecules in a microfluidic format is important for gene mapping technologies such as Direct Linear Analysis (DLA). We have recently shown that electric field gradients can be readily generated in a microfluidic device and the resulting field is purely elongational. We present a single molecule fluorescence microscopy analysis of T4 DNA (169 kbp) stretching in the electric field gradients in a hyperbolic contraction microchannel. In addition, we are able to selectively pattern a crosslinked gel anywhere inside the microchannel. With an applied electric field, DNA molecules are forced to reptate through the gel and they moderately stretch as they exit the gel. By placing a gel immediately in front of the hyperbolic contraction, we bypass "molecular individualism" and achieve highly uniform and complete stretching of T4 DNA.     

Transverse alternating field electrophoresis and applications to mammalian genome mapping

The transverse alternating field electrophoresis system is a pulsed field gel apparatus that has been used to separate DNA molecules that range in size from a few thousand to approximately 7 million base pairs. This apparatus uses a vertical gel and a simple electrode arrangement to produce electric fields that are uniform across all lanes of the gel. The velocity of identical molecules does not vary from lane to lane, and hence there is no distortion in the paths of the DNA. The performance of this system is illustrated here using the chromosomes from S. pombe and S. cerevisiae, and restriction enzyme digested mammalian DNA. The mobility of molecules up to 1100 kilobase pairs is linear with size and can be accomplished in overnight runs. Resolution of very large molecules requires electrophoresis for several days, but molecules from 200 to 7000 kilobase pairs can be separated on a single gel. This electrophoresis system has been used extensively in the construction of a physical map of human chromosome 21, and examples of this application are discussed.     

High-performance ultrathin layer agarose gel electrophoresis

Large scale, high-resolution DNA fragment analysis, such as genotyping, mapping and genetic profiling requires an affordable, fully automated high-throughput gel electrophoresis based separation device that enables rapid, high-performance analysis in a wide molecular weight range. In this article a novel approach is described that greatly enhances the productivity of DNA fragment analysis by automating the current manual procedure and also reducing the separation time and human intervention from sample loading to data analysis. The ultrathin layer, multilane, high-performance agarose gel electrophoresis system employs integrated scanning laser induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. The separation platform is fabricated in a way that the sieving matrix can be easily replaced in the separation cassette for each run. Visualization of the DNA fragments is accomplished by ‘in migratio' complexation during the electrophoresis process with ultra-sensitive fluorescent agents, also enabling real-time imaging and data analysis.     

Effect of electric field switching on the electrophoretic mobility of single-stranded DNA molecules in polyacrylamide gels

We have examined the effects of pulsed electric fields on the separation of single-stranded DNA molecules in polyacrylamide sequencing gels. Using different electric field pulsing regimens, the mobilities of single-stranded DNA molecules can be retarded or increased as compared to conventional electrophoresis. These results indicated that pulsed field techniques can be applied to gel electrophoresis of small single-stranded DNA molecules.     

Negative visualization of DNA in agarose gel by eosin Y and its related mechanism

We report here a sensitive and environmentally benign technique for the visualization of DNA by using eosin Y, which appeared as a transparent and colorless band under the opaque gel matrix background. As low as 0.2-0.6 ng of DNA can be visualized within 1 h, similar to that of the most commonly used ethidium bromide stain. Furthermore, the mechanism of the newly developed method was also described. According to the results, it can be concluded that the complex between DNA and eosin Y is mainly formed by the function of groove binding, but not intercalate itself into the double helix structure of DNA.     

Analysis of internucleosomal DNA fragmentation in apoptotic thymocytes by dynamic sieving capillary electrophoresis

The analysis, by slab gel electrophoresis, of internucleosomal DNA cleavage or laddering, characteristic of apoptosis in many cell systems, is labour intensive, difficult to automate and best only semi-quantitative. In this report we show that CE, using dilute solutions of hydroxyethylcellulose as a replaceable sieving matrix, can be applied to the relatively rapid analysis of DNA laddering in whole digests of apoptotic rat thymocytes. Also, using the sensitivity of laser-induced fluorescence detection and the highly sensitive nucleic acid stain YO-PRO-1, the CE method reported here can use 1000–2000 fold fewer cells than needed for traditional slab gel methods.     

Dynamic Expression of DNA Complexation with Self‐assembled Biomolecular Clusters

We report herein the implementation of a dynamic covalent chemistry approach to the generation of multivalent clusters for DNA recognition. We show that biomolecular clusters can be expressed in situ by a programmed self‐assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titration calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA‐binding clusters. Furthermore, we show that a chemical effector can be used to trigger DNA release through component exchange reactions.