Multiple-Locus Variable Number Tandem Repeat Analysis of Klebsiella pneumoniae: Comparison with Pulsed-Field Gel Electrophoresis.

AIM: To assess whether multiple-locus variable-number tandem repeat analysis (MLVA) could replace pulsed-field gel electrophoresis (PFGE) for genotyping of Klebsiella pneumoniae, this study was conducted to compare the typeability, discriminatory power, and concordance of these methods. MATERIALS AND METHODS: We used the nine variable number tandem repeat (VNTR) loci scheme to test its suitability for differentiating 114 ESBL-producing K. pneumoniae isolates collected from different clinical specimens in three hospitals of Tehran, Iran between April and December 2011. PFGE with XbaI was performed and the results were compared with those obtained by typing with MLVA. RESULTS: MLVA and PFGE yielded 44 and 64 types, respectively. Simpson's Diversity Index of MLVA was 0.896 (a 95% confidence interval of 0.850-0.942) and of PFGE was 0.962 (a 95% confidence interval of 0.943-0.981). Congruence between PFGE and MLVA was low. The adjusted Wallace coefficient of PFGE to MLVA was 0.946; however, MLVA was less able to predict PFGE type (32.5%). A range of 2-12 alleles was identified at VNTR loci with Simpson's diversity values between 0.017 and 0.818. CONCLUSION: MLVA is a PCR-based typing method and is much easier and more rapid in comparison to PFGE. These data indicate that the MLVA typing scheme used in this study is discriminative and reliable for typing of K. pneumoniae. However, optimization of the VNTR markers is required to improve the discriminatory power of the method.

Easy washing of lysed cell plugs for bacterial typing by pulsed-field gel electrophoresis using simple equipment.

We designed and tested equipment to wash plugs following cell lysis in pulsed-field gel electrophoresis (PFGE). Our system can wash 30 plugs simultaneously in 1h using 15L of Tris-EDTA buffer, which makes plug washing for PFGE less labor-intensive.

Nonmonotonous variation of DNA angular separation during asymmetric pulsed field electrophoresis.

Asymmetric pulsed field electrophoresis within crystalline arrays is used to generate angular separation of DNA molecules. Four regimes of the frequency response are observed, a low frequency rise in angular separation, a plateau, a subsequent decline, and a second plateau at higher frequencies. It is shown that the frequency response for different sized DNA is governed by the relation between pulse time and the reorientation time of DNA molecules. The decline in angular separation at higher frequencies has not previously been analyzed. Real-time videos of single DNA molecules migrating under high frequency-pulsed electric field show the molecules no longer follow the head to tail switching, ratchet mechanism seen at lower frequencies. Once the pulse period is shorter than the reorientation time, the migration mechanism changes significantly. The molecule reptates along the average direction of the two electric fields, which reduces the angular separation. A freely jointed chain model of DNA is developed where the porous structure is represented with a hexagonal array of obstacles. The model qualitatively predicts the variation of DNA angular separation with respect to frequency.

Report on overcoming the poor quality of Apai pulsotypes with a short review on PFGE for listeria monocytogenes.

Since listeriosis, caused by Listeria monocytogenes, is one of the important concerns of public health in Europe related to foodborne zoonoses, an efficient protocol for isolate typing is necessary when performing epidemiological studies. Three standardized PFGE protocols available for L. monocytogenes were briefly reviewed. Since observing a poor-quality of ApaI pulsotypes in our laboratory, enzymes from three different manufacturers were compared. The obtained pulsotypes showed that restriction digestion with ApaI from New England BioLabs should be complemented with a subsequent overnight incubation of PFGE plugs in TE buffer for better performance.

Increase in local protein concentration by field-inversion gel electrophoresis.

Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and nonspecific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein recovery efficiency. Here, we describe the enhancement of protein separation efficiency up to twofold in conventional one-dimensional PAG electrophoresis (1D PAGE), two-dimensional (2D) PAGE, and native PAGE by implementing pulses of inverted electric field during gel electrophoresis.

A systematic evaluation of the role of crystalline order in nanoporous materials on DNA separation.

The role of order within a porous separation matrix on the separation efficiency of DNA was studied systematically. DNA separation was based on a ratchet mechanism. Monodisperse colloidal suspensions of nanoparticles were used to fabricate highly ordered separation media with a hexagonal close-packed structure. Doping with a second particle size yielded structures with different degrees of disorder, depending upon the volume fraction of each particle size. Radial distribution functions and orientational order parameters were calculated from electron micrographs to characterize the scale of disorder. The peak separation distance, band broadening, and separation resolution of DNA molecules was quantified for each structure. DNA separation parameters using pulsed fields and the ratchet effect showed a strong dependence on order within the porous nanoparticle array. Ordered structures gave large separation distances, smaller band broadening and better resolution than highly disordered, nearly random, porous structures. The effect dominated these three parameters when compared to the effect of pore size. However, the effect of order on separation performance was not monotonic. A small, but statistically significant improvement was seen in structures with short range order compared to those with long range order.

Miniaturized system for rapid field inversion gel electrophoresis of DNA with real-time whole-gel detection.

Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications. But many aspects of DNA migration phenomena under pulsed field conditions are not well understood as compared with the more conventional situation where the electric field is held constant. A key reason for this deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (approximately 10-15 h) and the need to perform gel analysis by poststaining after completion of the run. Here we introduce an easy to build miniaturized slab gel apparatus that addresses these issues by enabling large DNA fragments up to 35 kb in length to be separated using field inversion gel electrophoresis (FIGE) in 60-90 min. The compact size of the device also allows the entire gel to be continuously monitored so that the separation processes can be imaged in real time using a high-resolution CCD camera. Arbitrary control over the applied voltage waveforms is achieved using a function generator interfaced with a high voltage amplifier. These capabilities allow us to probe the size dependence of fundamental physical parameters associated with DNA migration (mobility, diffusion, and separation resolution). These data reveal a surprising regime where separation resolution increases with DNA fragment size owing to a favorable interplay between mobility and diffusion scalings and highlight the important role of diffusion (a seldom quantified parameter). In addition to the practical benefit of separation times that are an order of magnitude faster than conventional instruments, the results of these studies provide a previously unavailable rational basis to identify optimal separation conditions and contribute new insights toward understanding the underlying physical processes that govern DNA electrophoresis in pulsed fields.

An automated sample preparation system with mini-reactor to isolate and process submegabase fragments of bacterial DNA.

Existing methods for extraction and processing of large fragments of bacterial genomic DNA are manual, time-consuming, and prone to variability in DNA quality and recovery. To solve these problems, we have designed and built an automated fluidic system with a mini-reactor. Balancing flows through and tangential to the ultrafiltration membrane in the reactor, cells and then released DNA can be immobilized and subjected to a series of consecutive processing steps. The steps may include enzymatic reactions, tag hybridization, buffer exchange, and selective removal of cell debris and by-products of the reactions. The system can produce long DNA fragments (up to 0.5 Mb) of bacterial genome restriction digest and perform DNA tagging with fluorescent sequence-specific probes. The DNA obtained is of high purity and floating free in solution, and it can be directly analyzed by pulsed-field gel electrophoresis (PFGE) or used in applications requiring submegabase DNA fragments. PFGE-ready samples of DNA restriction digests can be produced in as little as 2.1 h and require less than 10(8) cells. All fluidic operations are automated except for the injection of the sample and reagents.

Investigating DNA migration in pulsed fields using a miniaturized FIGE system.

PFGE is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here, we present a simple miniaturized FIGE apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 h using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N(-0.5) over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively.

[Pulsed field gel electrophoresis: theory, instruments and applications].

Pulsed Field Gel Electrophoresis (PFGE) is a powerful technique for the fractionation of high molecular weight DNAs ranging from 10 kb to 10 Mb in size. PFGE separates DNA molecules in agarose gel by subjecting them to electric fields that alternate ("pulsate") in two directions. This technology plays a key role in the modern genomics as it allows manipulations of the DNA of whole chromosomes or their large fragments. In this review we discuss: 1) the theory behind PFGE, 2) different instruments based on the principle of pulsed field, their advantages and limitations, 3) factors affecting the mobility of DNA in PFGE gels, 4) practical applications of the technique.

Application of microchip-capillary electrophoresis and pulsed electrochemical detection to the analysis of biologically relevant phenolic compounds.

In this report, the most recent results regarding the use of microchip-capillary electrophoresis and pulsed electrochemical detection are reviewed. This article is particularly focused on the analysis of three groups of compounds: phenolic contaminants, phenolic acids, and phenolic antioxidants. Background information and a brief discussion covering other related analytical strategies are also included.

A method for reproducibly preparing synthetic nanopores for resistive-pulse biosensors.

There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for biomedical analytes. Analytes detected with prototype artificial-nanopore biosensors include drugs, DNA, proteins, and viruses. This field is, however, currently in its infancy. A key question that must be addressed in order for such sensors to progress from an interesting laboratory experiment to practical devices is: Can the artificial-nanopore sensing element be reproducibly prepared? We have been evaluating sensors that employ a conically shaped nanopore prepared by the track-etch method as the sensor element. We describe here a new two-step pore-etching procedure that allows for good reproducibility in nanopore fabrication. In addition, we describe a simple mathematical model that allows us to predict the characteristics of the pore produced given the experimental parameters of the two-step etch. This method and model constitute important steps toward developing practical, real-world, artificial-nanopore biosensors.

Pulsed-field gel electrophoresis.

This protocol describes pulsed-field gel electrophoresis (PFGE), a method developed for separation of large DNA molecules. Whereas standard DNA gel electrophoresis commonly resolves fragments up to approximately 50 kb in size, PFGE fractionates DNA molecules up to 10 Mb. The mechanism driving these separations exploits the fact that very large DNA molecules unravel and "snake" through a gel matrix, and such electrophoretic trajectories are perturbed in a size-dependent manner by carefully oriented electrical pulses. PFGE has enabled the rapid genomic analysis of microbes and mammalian cells, and motivated development of large-insert cloning systems such as bacterial and yeast artificial chromosomes. As such, this protocol includes descriptions of two types of PFGE instrumentation (not commercially available), along with detailed instructions for their operation. Additionally, this protocol provides basic instructions for the preparation of intact chromosomal DNA from several types of organisms. PFGE takes 2-3 days, excluding sample preparation.

Capacitively-induced pulsed-field gel electrophoresis: a novel method for DNA separation.

Present instruments used in pulsed field and conventional gel electrophoresis, encounter a number of real and conceived difficulties such as electrical hazard, ground leak current, electrical noises, formation of gas bubbles at the metal electrodes and production of Joule heat in the buffer-gel system. To overcome the above-mentioned problems a novel electrophoresis unit based on capacitively-induced pulsed field was designed and tested in which the applied high voltage is decoupled from the electrolyte (buffer-gel system). In order to achieve a higher performance, the primary pulse generator which has been fabricated to apply for capacitively-induced pulsed field electrophoresis was equipped with a modulating frequency pulse generator which produced a combination of low and high frequency waves. The newly designed electrophoresis unit was able to resolve the Lambda DNA fragments so that seven bands with an acceptable resolution were observed. By increasing the run time both the depletion of molecules from the wells and the resolution of the bands improved compare to the patterns obtained via conventional electrophoresis.

Description of an ultrathin multiwire proportional chamber-based detector and application to the characterization of the Spraguea lophii (Microspora) two-dimensional genome fingerprint.

Multiwire proportional chamber is a useful technology to build detectors that supersede the lack of interactivity of autoradiography in molecular biology experiments. Some drawbacks still limited the diffusion of existing instruments in biological laboratories. The major competitors are storage phosphor imaging systems. The simplified description of a radio-chromato-imager prototype (RCI) based on an original ultrathin multiwire proportional chamber is presented. It combines the advantage of the different existing technologies to present competitive properties in terms of efficiency, spatial resolution, robustness, manipulation easiness and production cost. Application of the RCI detector to molecular biology was performed by the analysis of karyotype and restriction display two-dimensional pulsed-field gel electrophoresis (KARD 2-D PFGE) data which are used to describe small eukaryotic genome structures. The comparative analysis with autoradiography was performed with the PDQuest software on Spraguea lophii (Microspora) genome fingerprints. The spot detection procedure applied to the different images leads to a similar conclusion considering the genome structure of S. lophii which appeared to be composed of 15 chromosomes for 13 karyotypic bands (200-880 kbp).

Mechanisms of DNA separation in entropic trap arrays: a Brownian dynamics simulation.

Using Brownian dynamics simulations, we study the migration of long charged chains in an electrophoretic microchannel device consisting of an array of microscopic entropic traps with alternating deep regions and narrow constrictions. Such a device has been designed and fabricated recently by Han and Craighead [Science 288 (2000) 1026] for the separation of DNA molecules. Our simulation reproduces the experimental observation that the mobility increases with the length of the DNA. A detailed data analysis allows to identify the reasons for this behavior. Two distinct mechanisms contribute to slowing down shorter chains. One has been described earlier by Han and Craighead [Science 288 (2000) 1026]: the chains are delayed at the entrance of the constriction and escape with a rate that increases with chain length. The other, actually dominating mechanism is here reported for the first time: some chains diffuse out of their main path into the corners of the box, where they remain trapped for a long time. The probability that this happens increases with the diffusion constant, i.e., the inverse chain length.

Nafion membrane electrophoresis with direct and simplified end-column pulse electrochemical detection of amino acids.

A novel electrophoresis technique, in which the separation column was replaced by a strip of Nafion membrane (5.0 cm x 0.20 mm x 0.25 mm), was developed for the separation of an amino acid mixture (glycine, asparic acid and lysine), followed by quadruple-pulse electrochemical detection. Nafion membrane contains hydrophilic pores (10-20 A and 50-60 A in size) acting as very narrow electrophoresis channels. The fixed-charge sites (-SO(3) (-)) on the hydrophilic pore surface provide a strong charged background. A platinum disk electrode (0.90 mm inner diameter) was employed as the detection electrode and the electrophoresis cathode was used as the quasi-reference and counter electrode for the end-column electrochemical detector, without decoupler. Under optimized conditions the mixture of amino acids could be separated at a voltage of only 90 V with a detection limit of 10(-7) M, indicating that Nafion membrane electrophoresis is a potentially attractive technique for the separation of small organic molecules or ions.

Improved resolution with microchip-based enhanced field inversion electrophoresis.

We present an improvement of the field inversion electrophoresis (FIE) method in which the passage of sample such as DNA back and forth within a short length of a microchannel can provide a similar resolution to that of a significantly longer microchannel. In constant field FIE the application of an alternating potential (e.g., +/- V) over short periods of time (e.g., several Hz) can provide enhanced separations of DNA fragments. In contrast, the present method consists of a series of separations, each of much longer duration, under high and low fields in such a way that the resolution is enhanced. This method is readily modeled and allows improved resolution to be obtained from extremely short microchannels (e.g., 8 mm) while requiring relatively low applied voltages (e.g., less than 600 V). An additional advantage is that this method can allow for the same equipment to be used in a rapid, low-resolution mode or in a slower, high-resolution mode through what might be referred to as an automated "zoom" capability. We believe that this method may facilitate the integration of microfluidic devices and microelectronic devices by allowing these devices to be of a similar small scale (< 1 cm).

Simple circuit to improve electric field homogeneity in contour-clamped homogeneous electric field chambers.

We redesigned contour-clamped homogeneous electric field (CHEF) circuitry to eliminate crossover distortion, to set identical potentials at electrodes of each equipotential pair and to drive pairs with transistors in emitter follower stages. An equipotential pair comprised the two electrodes set at the same potential to provide electric field homogeneity inside of the hexagonal array. The new circuitry consisted of two identical circuits, each having a resistor ladder, diodes and transistors. Both circuits were interconnected by diodes that controlled the current flow to electrodes when the array was energized in the 'A' or 'B' direction of the electric field. The total number of transistors was two-thirds of the total number of electrodes. Average voltage deviation from potentials expected at electrodes to achieve a homogeneous electric field was 0.06 V, whereas 0.44 V was obtained with another circuit that used transistors in push-pull stages. The new voltage clamp unit is cheap, generated homogeneous electric field, and gave reproducible and undistorted DNA band patterns.

An inexpensive sample mold for pulsed-field electrophoresis.

Specimens for pulsed-field gel electrophoresis (PFGE) are formed using a plug mold. We report a technique which uses a disposable polyethylene pipette to prepare our specimen plug. We also report a convenient technique to handle portions of the plug used for the typical PFGE manipulations.