Separation Of DNA Restriction Fragments Research Articles

DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds.

Low-viscosity polymer solutions have potential for double-stranded (ds) DNA separations in micrototal analysis systems (micro-TAS). In this paper, we report dilute, low-viscosity hydroxypropylmethylcellulose-50 (HPMC-50, 11.5 kDa) solutions containing polyhydroxy additives as separation media. Predominant operational variables, such as applied electric field strength, fluorescent intercalator (YOPro-1) concentration, polymer concentration, and additive concentration, are thoroughly investigated. Fast (within 170 s) and excellent separation of DNA restriction fragments ranging in size from 72 to 1353 base pairs (bp) is achieved in a 30 mm length channel of polymethylmethacrylate (PMMA) microchips at an electric field strength of 300 V/cm, by introducing 8% mannitol, 8% glucose or 10% glycerol additives into a 2% HPMC-50/1 x Tris-borate-EDTA (TBE) solution. The low-viscosity (40 cP) matrix formulation provides both coating of the microchannels and separation of DNA in one step. The performance in the solution surpasses that in highly concentrated HPMC-50 solution. In addition, separation using 1xTris-EDTA buffer in the 2% HPMC-50 matrix containing polyhydroxy additives also exhibits a notably increased performance. This is presumably due to formation of hydrogen-bonding interactions of polyhydroxy additives with HPMC-50 matrix and DNA so as to increase the coupling interactions between matrix and DNA molecules during electrophoresis. The result reflects that boric acid is not a prerequisite in polyhydroxy-enhanced HPMC-50 solution for separation.

Les marqueurs moléculaires pour l’étude épidémiologique des infections à Legionella pneumophila

Purpose. – Legionnaires’ disease is due to the inhalation of contaminated aerosols. The identification of the source of contamination in the aquatic environment is necessary to prevent the occurrence of new cases. A comparative study of clinical and environmental isolates is the basis of epidemiological investigations. Current knowledge and key points. – Genotypic methods are now mainly used to compare bacterial strains. Some of these methods are based on the electrophoretic separation of DNA restriction fragments. When electrophoretic profile are complex, some fragments can be visualized after hybridization (ribotyping). Large-sized fragments can be separated by pulsed-field gel electrophoresis. Other techniques are based on gene amplification, such as AP-PCR. This technique is easy to perform but its discriminatory power and reproducibility are lower. Some procedures are combining enzymatic cleavage and gene amplification. Finally methods based on the nucleotide sequence analysis of some genes are being evaluated. Future prospects and projects. – Techniques enabling the rapid comparison of various Legionella isolates will permit a quick detection of outbreaks and contribute to the identification of the source of contamination.

Meat speciation by restriction fragment length polymorphism analysis using an α-actin cDNA probe

Classical DNA fingerprinting is based on separation of DNA restriction fragments by electrophoresis and hybridisation to nucleic acid probes containing repetitive nucleotide sequences. The use of such mini- or micro-satellite probes tends to yield patterns specific to an individual rather than to a species, hence their value in forensic analysis but general unsuitability for meat speciation. In the present study, a cDNA probe based on conserved sequences contained in members of the actin multigene family has been evaluated for potential application in meat speciation. Genomic DNA was extracted from muscle and digested with BamHI before electrophoresis and hybridisation to a murine α-actin cDNA probe. Beef, pork, lamb, horse, chicken and fish DNA restriction fragments formed characteristic ‘fingerprints’ which were reproducible and varied sufficiently to allow discrimination even between closely-related species. However no major differences were seen between individuals of the same breed or between different breeds within a species. When DNA obtained from fresh tissue and also from meat heated at 120 °C was analysed, the gel patterns were essentially the same. An attractive feature of this approach is that it employs a single cross-reacting probe and set of conditions, and gives different patterns with all species so far studied. This simplicity suggests applications in meat speciation or related areas of biology.

High-Resolution Separation of DNA Restriction Fragments Using Capillary Electrophoresis with Near-IR, Diode-Based, Laser-Induced Fluorescence Detection

The near-IR dye thiazole green (TAG) was used as a monomeric nuclear staining dye for the low-level detection of DNA restriction fragments separated via high-performance capillary electrophoresis with near-IR laser-induced fluorescence detection. TAG possessed an absorption maximum at 735 nm and an emission maximum at approximately 765 nm and, in the presence of dsDNAs, showed a fluorescence enhancement ratio of approximately 102, with a binding constant to dsDNAs determined to be 6.1 × 106 M-1. The high-resolution separation of the HaeIII restriction digest of ΦX174 was carried out using capillary electrophoresis on the native, ethidium bromide-stained, and TAG-stained DNA fragments. The TAG-stained DNA fragments resulted in higher plate numbers compared to the native and EtBr-stained restriction fragments as well as enhanced resolution; however, the 271/281 fragments could not be resolved using these CE conditions. To investigate the detection sensitivity of the TAG-stained DNA in capillary electrophore...

Evaluation of the genetic diversity among maize lines (Zea mays L.) by DNA fingerprinting analysis

Informativo DNA fingerprint profiles of eight homozygotic maize lines were obtained by the electrophoretic separation of DNA restriction fragments and the ir hybridization with the minisatellite probe R18.1. The analysis of the bandsharing frequencies allowed to identify all the lines and to estimate the genetic distances between them. The relationship obtained by DNA fingerprinting analysis of the eight inbreed lines was highly consistem with the ir genetical origin.

Capillary gel electrophoretic separation of DNA restriction fragments in a discontinuous buffer system

Separation of single and double stranded DNA molecules by capillary gel electrophoresis has a rapidly growing interest. Similar to the polycrylamide slab gel based separation methods, in capillary gel electrophoresis, the paek/bans spacing usually decreases with the increasing size/length of the DNA molecule. Additionally, employing the regularly used Tris-borate buffer system, fronting peaks are often obtained. By the application of an electrolyte step gradient during capillary gel electrophoretic separation of dsDNA molecules, the apparent peak shape can be improved and the required analysis time decreased.

The effects of polymer properties on DNA separations by capillary electrophoresis in uncross-linked polymer solutions.

Low-viscosity, aqueous solutions of hydrophilic linear polymers have been shown to be useful for the separation of DNA restriction fragments by capillary electrophoresis (CE). However, the choice of polymer type, size, and concentration remains largely empirical, because the mechanism of high-field electrophoretic DNA separations in polymer solutions is not well understood. To assist in elucidating the mechanism of DNA separation, we experimentally investigated the effects of polymer properties such as stiffness (persistence length), average molecular mass, polydispersity, and hydrophilicity on the separation of DNA ranging from 72 bp to 23 kbp. This was accomplished by comparing the results of DNA separations obtained by counter-migration CE in dilute and semidilute solutions of linear polyacrylamide (PAA), hydroxyethyl-cellulose (HEC), and hydroxypropylcellulose (HPC) polymers of several different average molecular masses.

Capillary Electrophoretic Separation of DNA Restriction Fragments in Mixtures of Low- and High-Molecular-Weight Hydroxyethylcellulose

Previous studies (Barron et al., 1993, 1994) have shown that dilute aqueous solutions of hydroxyethylcellulose (HEC) polymers provide an excellent alternative to gel-based DNA separation media for capillary electrophoresis (CE) of DNA restriction fragments (72−23 130 base pairs). DNA separation by CE in HEC solutions is strongly influenced by the average HEC molecular weight as well as by the HEC concentration in the electrophoresis buffer. Here we describe a systematic investigation of the effects of a mixture of low- and high-molecular weight HEC polymers, over a range of concentrations, to form a DNA separation medium with a broad range of polymer chain lengths. Our results show that, relative to separation media containing solely low-molecular-weight or high-molecular-weight HEC polymers, the mixed polymer solutions provide superior separation over the DNA size range of interest, while providing a five-fold viscosity reduction. The addition of a very small amount of high-molecular-weight HEC to a solution of low-molecular-weight HEC leads to a significant improvement in the separation of larger DNA fragments (>603 base pairs), while retaining resolution of smaller DNA fragments. The consequences of these results on the proposed mechanism of DNA separation in un-cross-linked polymer solutions are discussed.

Comparative study of non-porous anion-exchange chromatography, capillary gel electrophoresis and capillary electrophoresis in polymer solutions in the separation of DNA restriction fragments

HPLC using a 100-mm column packed with a non-porous anion exchanger gave high-resolution separations of DNA restriction fragments up to 8000 base pairs (bp). High-resolution separation of DNA restriction fragments up to 12 000 bp was achieved using capillary gel electrophoresis (CGE) and capillary electrophoresis (CE) in entangled polymer solutions. These methods were compared with respect to their performance and efficiencies in the resolution of DNA restriction fragments. The resolving power of CGE is higher than those of the other techniques. The 50–100 bp resolution of DNA fragments up to 500 bp was realized using HPLC, 50 bp resolution using CE in entangled polymer solutions and 10 bp resolution using CGE. The plate number that was achieved with CGE of 3 · 10 6 plates/m was higher than those of HPLC of 1 · 10 6 and CE in polymer solutions of 7 · 10 5 plates/m. The advantages and limitations of HPLC and capillary electrophoretic techniques are discussed.

High-Speed Parallel Separation of DNA Restriction Fragments Using Capillary Array Electrophoresis

A new method for performing high-speed, high-throughput sizing of DNA has been developed. Samples containing DNA restriction fragments between 70 and 10,000 base pairs in length are electrophoretically separated using capillary arrays in ∼20 min and detected with high sensitivity using a fluorescence detection system. The separations of ΦX174/HaeIII fragments are performed on an array of seven 100-μm i.d., 350-μm o.d. capillaries using replaceable hydroxyethyl-cellulose solutions as the sieving medium. The fragments are fluorescently labeled using ethidium bromide in the running buffer and detected with a laser-excited, confocal-fluorescence scanner. The limit of detection is ∼1 pg of DNA per band and separations can be detected injecting DNA samples as dilute as 0.1 ng/μl. Samples were electrokinetically injected from a cassette of microcentrifuge tubes using a format that will facilitate the application of capillary electrophoresis to separations in molecular biology. The feasibility of extending this technique to lower DNA concentrations and to ∼100 capillary arrays is discussed.

High-resolution separation of DNA restriction fragments by capillary electrophoresis in cellulose derivative solutions

The performance and the efficiency of several cellulose derivatives as a molecular sieving agent for the capillary electrophoretic separation of DNA restriction fragments were investigated. All fragments up to 12 000 base pairs (bp) in the 1-kbp DNA ladder were resolved using linear polyacrylamide-coated capillaries filled with a buffer solution containing 0.5% cellulose derivative and the separation was completed within 17 min. High-concentration (0.7%) cellulose derivative solutions are effective for the complete separation of small fragments (50–1000 bp) of a HincII and a HaeIII digest of ΦX174 DNA. A plate number of 0.5–1·10 6 plates per metre was achieved. The migration time and the resolution of DNA fragments were manipulated by varying several parameters, such as the size (viscosity) and the concentration of cellulose derivatives and the applied field strength. Some guidelines are presented for choosing these parameters, depending on the size of the DNA fragments being separated.

Separation of DNA restriction fragments by polymer-solution capillary zone electrophoresis: Influence of polymer concentration and ion-pairing reagents

To achieve sensitive and fast separations of the DNA fragments, polymer-solution capillary zone electrophoresis (PS-CZE) is examined. A systematic study of the influence of the linear polymer concentration on the separation of restriction fragments indicates that the larger DNA fragrments are resolved with greater efficiency by using a lower concentration of the polymer, while the smaller ones are better resolved with a higher polymer concentration, when added to the buffer. A specific concentration of the polymer provides a certain limit of dynamic “porosity” (sieving), which is appropriate for a given range of the DNA fragments. Several ion-pairing reagents are examined here to improve resolutions of both small and large DNA fragments in PS-CZE. These reagents cause no interactions between the ammonium cations (reagent) and the silanol groups (capillary surface). However, they interact with the linear polymer, perhaps by hydrophobic functions, and change its physical properties, such as dynamic viscosity and sieving ability. Enhanced retention (interactions) of the DNA fragments is observed by increasing the ion-pairing contents in the polymer solution. The increased peak retention strongly suggests presence of interactions between the reagent and the DNA fragments. Different ion-pairing reagents produce different degrees of peak retentions. The interactions results from relatively greater hydrophobic interactions and smaller ionic interactions between the reagent and the polynucleotides. The study of ethidium bromide (EdBr) additon in PS-CZE indicates that a small concentration of EdBr reduces peak widths and decreases retention of all the fragments, but has little influence on the electroosmotic flow. However, a larger EdBr concentration results in broader peak widths and causes little change in their retention. Ion-pairing and intercalating reagents interact with the DNA fragments by different mechanisms. An anion-pairing agent appears to interact predominantly by hydrophobic interactions with the purine and pyrimidine bases, while EdBr intercalates with G:C base pairs and alter the chain length. Satisfactory separations of both larger and smaller fragments can be achieved by adding an appropriate ion-pairing reagent along with the linear polymer to the buffer. Critical concentrations of the linear polymer and the ion-pairing reagent must be determined in order to achieve satisfactory separations for a given range (size) of the DNA fragments.

Capillary electrophoresis of DNA restriction fragments with solutions of entangled polymers.

Separation of DNA restriction fragments in dilute buffer solutions of network forming polymers such as linear polyacrylamide (PAA), hydroxyethyl cellulose (HEC) and polyvinylalcohol (PVA) in phosphate buffer were investigated. PVA in buffers already became inhomogeneous after a few separations with resultant deterioration of resolution. Hydroxylic polymers, capable of forming suitable networks in buffers, are strongly adsorbed to fused silica surfaces suppressing the electroosmotic flow. These polymers could be applied as surface modifiers in capillaries, filled with buffer media containing other polymers such as PAA. Suppression of the electroosmotic flow by adsorbed HEC at pH 7 was slightly more effective than with PVA but the former coating was less stable due to weaker binding to the fused silica surface. Good separations of restriction fragments could be achieved with solutions of PAA or HEC as separation media after surface pretreatment of fused silica capillaries by either PVA rinsing or coating according to Hjertén.

Enhanced separation of DNA restriction fragments by capillary gel electrophoresis using field strength gradients

The effect of electric field strength gradients on the separation of DNA restriction fragments was investigated. As reported in our earlier work, the mobility of different size double-stranded DNA molecules is a function of the applied electric field which suggests that the use of a nonuniform (time varying) electric field may increase the resolving power. We demonstrate that in capillary gel electrophoresis enhanced separation of DNA restriction fragments up to 1353 bases pairs (bp) in size can be achieved by employing the field strength gradient method

Effect of temperature on the separation of DNA restriction fragments in capillary gel electrophoresis

Two different separation modes were used in high-performance capillary gel electrophoresis to study the effect of temperature on the separation of DNA restriction fragments, isoelectrostatic (use of constant applied electric field) and isorheic (use of constant current). In both instances, the migration properties and resolution of the DNA molecules were studied as a function of column temperature between 20 and 50°C. In the isoelectrostatic separation mode, the migration time and resolution decrease as temperature increases. In the isorheic separation mode, increasing the column temperature results in a maximum migration time for all of the DNA fragments and shows a maximum resolution for the lower molecular weight fragments (<200 kilodalton).

Genome scanning of higher eukaryotes by two-dimensional DNA typing using micro- and minisatellite core probes

Electrophoretic separation of DNA restriction fragments in two dimensions according to two independent criteria, i.e., size and basepair sequence, allows all restriction fragments of DNA molecules of up to 5 million bp, such as the Escherichia coli genome, to be resolved in a single gel. For more complex genomes, hybridization analysis of the two-dimensional separation patterns is necessary to obtain interpretable data. Here we describe a detailed protocol for two-dimensional DNA typing of mammalian (human and cattle) and plant (tomato) genomic DNA using micro- and minisatellite core probes. Spot patterns comprising 100–800 spots were obtained. This method of simultaneously scoring hundreds of genetic loci has various applications e.g., in mapping genomes, defining DNA markers for hereditary traits by genetic linkage analysis, and detecting somatic instability.

Separation of DNA restriction fragments by capillary electrophoresis using coated fused silica capillaries.

The abilities of several different capillary electrophoresis techniques to separate DNA restriction fragments up to 23,000 bp were investigated. Methods employing electroosmotic flow in an untreated silica capillary were found to provide, at best, only partial resolution of the 23 fragments in a 1-kbp DNA ladder. By coating the inner walls of a silica capillary with poly(acrylamide) and filling these capillaries with buffers containing methylcellulose as a sleving medium, all fragments in the 1-kbp DNA ladder were separated. In addition, this technique facilitated the separation of the very large fragments in a lambda DNA-HindIII digest. Optimum resolution was obtained at low separation potentials using buffers containing at least 0.5% methylcellulose. The performance of this technique, i.e., resolution and quantitation, make capillary electrophoresis a powerful complement to slab gel electrophoresis and may make it a preferred alternative to both agarose gel electrophoresis and HPLC for applications such as the confirmation of plasmid integrity.

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacryl- amide with low amounts of crosslinking agent ( i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels ( e.g. 5%C), permitted high efficiency separations of double stranded DNA fragments up to 12 000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots ( i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacryl- amide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.

Separation of DNA restriction fragments by high-performance ion-exchange chromatography on a non-porous ion exchanger

Separation of DNA restriction fragments by high-performance ion-exchange chromatography on a non-porous ion exchanger

Separation of DNA restriction fragments by high-performance ion- exchange chromatography on a non-porous ion exchanger

Separation of DNA restriction fragments by high-performance ion- exchange chromatography on a non-porous ion exchanger