Separation Of Large DNA Fragments Research Articles

Recent application developments in genomic research using the AdvanCE™ FS capillary electrophoresis platform

Central to genomic research is the analysis of nucleic acids, which requires versatile, sensitive and high throughput technologies that can deliver accurate results in a short time. Reported herein are recent developments for sizing and/or quantifying nucleic acids (dsDNA and RNA) on a parallel capillary electrophoresis platform with LED-based fluorescence detection (AdvanCE™ FS system). This flexible multi-capillary platform has been employed for applications such as SSR, gDNA analysis, RNA analysis, quantification of next generation sequencing (NGS) libraries, and the separation and sizing of large cDNA from Populus spp,This poster will discuss recent validation of the platform for a subset of these applications in detail. The AdvanCE™ FS system successfully scored all SSR samples with resolution as low as 2 bp. Assessment of the quality/concentration of gDNA is also demonstrated with a new separation matrix, for separation of large DNA fragments. Separation and sizing of large cDNA was also demonstrated with good resolution and accuracy. A method for rapid and sensitive detection of total RNA concentration and quality will be discussed, as well as the sizing and quantification of NGS libraries.The AdvanCE™ FS platform offers rapid separation and ample resolution with excellent sensitivity and dynamic range, to benefit a variety of applications in genomic research.

Bioanalysis in structured microfluidic systems

Microfluidic and lab-on-a-chip devices have attracted widespread interest in separation sciences and bioanalysis. Recent designs in microfluidic devices extend common separation concepts by exploiting new phenomena for molecular dynamics on a length scale of 10 mum and below, giving rise to novel manipulation tools and nonintuitive phenomena for microseparations. Here, we focus on three very recent developments for bioseparations based on tailored microfluidic systems: Single cell navigation, trapping and steering with subsequent on-chip lysis, protein separation and LIF detection (Section 3.1), then we report dielectrophoretic trapping and separation of large DNA fragments in structured microfluidic devices (Section 3.2). Finally, a paradoxial migration phenomenon based on thermal fluctuations, periodically arranged microchannels and a biased alternating current electric field is presented in Section 3.3.

Bridging the gap between molecular epidemiologists and evolutionists

Molecular epidemiology designates the various molecular methods that aim to identify the relevant units of analysis of pathogens involved in transmissible diseases: species, subspecies, strains, clones and genes of interest. It is frequently based on an empirical approach. I advocate that evolutionary concepts enrich this discipline considerably and should be considered as an integral part. In turn, the experience and questioning of field experts are crucial to evolutionists who use transmissible diseases as models. A molecular epidemiology aim gives evolutionary studies a practical goal, putting a stop to approaches that are overly speculative.

Rapid Separation of Large DNA Fragments

Rapid Separation of Large DNA Fragments

Sparsely cross-linked "nanogels" for microchannel DNA sequencing.

We have developed sparsely cross-linked "nanogels", sub-colloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel DNA sequencing matrices for capillary electrophoresis. The presence of covalent cross-links affords nanogel matrices with enhanced network stability relative to standard, linear polyacrylamide (LPA), improving the separation of large DNA fragments. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and N,N-methylenebisacrylamide (Bis). In order to retain the fluidity necessary in a replaceable polymer matrix for capillary array electrophoresis (CAE), a low percentage of the Bis cross-linker (< 10(-4) mol%) was used. Nanogels were characterized by multiangle laser light scattering and rheometry, and were tested for DNA sequencing by CAE with four-color laser-induced fluorescence (LIF) detection. The properties and performance of nanogel matrices were compared to those of a commercially available LPA network, which was matched for both weight-average molar mass (Mw) and extent of interchain entanglements (c/c*). Nanogels presented in this work have an average radius of gyration of 226 nm and a weight-average molar mass of 8.8 x 10(6) g/mol. At concentrations above the overlap threshold, nanogels form a clear, viscous solution, similar to the LPA matrix (Mw approximately 8.9 x 10(6) g/mol). The two matrices have similar flow and viscosity characteristics. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, a substantially longer read length ( approximately 63 bases, a 10.4% improvement) is obtained with the nanogel matrix at 98.5% accuracy of base-calling. The nanogel network provides higher-selectivity separation of ssDNA sequencing fragments longer than 375 bases. Moreover, nanogel matrices require 30% less polymer per unit volume than LPA. This is the first report of a sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrices matched for Mw and extent of interchain entanglements.

Strategies for the separation of polyelectrolytes based on non-linear dynamics and entropic ratchets in a simple microfluidic device

We perform Monte Carlo simulations of an existing electrophoretic microchannel device used for the size separation of large DNA fragments. This device is normally operated with a constant (dc) driving field. In contrast, we consider the case of a varying (ac) driving field, in the zero-frequency limit. We find that a time-asymmetric pulse can yield interesting migration regimes, in particular bidirectional transport for different molecular sizes. We also study a spatially asymmetric version of the device and show that it can rectify unbiased but non-equilibrium molecular motion, in agreement with previous predictions for entropic ratchets. Finally, at finite frequency we uncover a resonance for the molecular velocity in the channel which could lead to improved performance.

Rapid Separation of Large DNA Fragments

Bacterial fingerprinting involving pulsed-field gel electrophoresis (PFGE) attracts special attention of the researchers as a promising method for medical diagnostics (1–7). Bacterial fingerprinting requires long DNA chains in a wide range of size from 24 to 900 kb to be resolved. In certain cases, DNA should be separated in several size ranges. For example, a high quality of DNA fragment resolution in three size ranges—from 225 to 900 kb (10 bands), from 240 to 700 kb (about 10 bands), and from 50 to 500 kb (14 bands)—is required for detecting Bordetella pertussis DNA fragments. Another example is diagnostics of Campilobacter upsalienis, requiring resolution of DNA fragments in two ranges, from 50 to 150 and from 45 to 775 kb. The clampet homogeneous electrical field (CHEF) technique and equipment DRII-III by Bio-Rad Laboratories are conventionally used for PFGE fingerprinting. Such PFGE session takes usually 18 to 24 h. Its duration, necessary for sufficient separation of the fragments, is one of the main disadvantages preventing wide application of PFGE fingerprinting. Recently, methods for rapid separation of large DNA fragments were reported (8, 9, 18); however, although ingenious, they are too far from being applied practically. We are proposing a simple yet efficient method allowing large DNA fragments to be separated fourto five-fold more rapidly compared with the CHEF-based technique. Our method involves a modification of the

Pulsed-field capillary electrophoresis separation of large DNA fragments.

Field-inversion capillary electrophoresis (FICE) provides many of the same benefits as the more familiar, field-inversion gel electrophoresis (FIGE). In the capillary format, field inversion allows size separation of linear dsDNA into the megabase region. Whereas FIGE separation of megabase pair (Mb) DNA takes 24 h or more, the most rapid FICE separations are complete within 4 min (,). As with any capillary electroseparation, the technique is rapid and provides high resolution, but is analytical scale only. Preparative scale use of capillary electrophoresis (CE) is not very practical, although apparatus capable of the collection of CE fractions have been developed ().

Capillary electrophoresis of DNA restriction fragments: effect of polymer properties.

The mechanism of DNA separation by dilute polymer solutions in capillary electrophoresis is not well understood. To provide information on the effect of polymer properties on DNA separations, four polymers that differ in size, shape and stiffness were examined. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp - 23,130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp; star-poly(ethylene oxide) and linear poly(ethylene oxide) provide separation of fragments to 1353 bp.

Ultrafast high resolution separation of large DNA fragments by pulsed‐field capillary electrophoresis

Pulsed-field capillary electrophoresis (PFCE) in buffers containing ultradilute polymer solutions is used to separate long chain dsDNA in less than 4 min. Separations are shown to work with chain lengths below 10 kbp and greater than 1.5 Mbp. Several pulse protocols have been examined. If running time is to be minimized, a field inversion with higher peak amplitude in the forward direction than in the reverse, but with equal pulse durations, provides the best resolution. Other protocols can provide higher resolution, but only with longer running times.

11] Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) of agarose gels enables the reproducible separation of large DNA fragments. In concept, PFGE is an extension of conventional electrophoresis, in which two alternating (or pulsed) electric fields are used instead of the traditional single static field. Separation occurs when these fields are oriented at an obtuse angle to one another. In a pulsed-field gel, the end of each molecule migrates in a new direction, with each change of the electric fields. The DNA molecules, thus, migrate through the agarose matrix in a zigzag motion. The tardiness of the larger molecules, in turning corners (e.g., in PFGE) or in running forward and backward [e.g., in field-inversin gel electrophoresis (FIGE)], separates them from the smaller size fragments. The effectiveness of PFGE, however, is not limited to the separation of very large DNA molecules. PFGE can improve the resolution of DNA molecules of only a few hundred bases and permits separation up to 12,000 kilobase pairs (kb). A number of models and theories have been proposed to explain some of the more complex behavior of DNA molecules in PFGE. However, biologists rarely need to consult these physical models or equations for practical PFGE applications. This chapter discusses the optimum PFG electrophoretic conditions for the separation of DNA fragments from 1 to 6000 kb.

Separation of large DNA fragments by capillary electrophoresis under pulsed-field conditions.

Capillary zone electrophoresis (CZE), using an entangled polyacrylamide solution, was applied to large DNA samples under pulsed-field conditions. Highly efficient separations were achieved under biased sinusoidal field and field-inversion pulsing regimes. The separations that were obtained with 8.3-48.5 kb lambda DNA standards and 48.5 kb-1 Mb lambda DNA concatamers (modified with ethidium bromide) clearly demonstrate a dramatic improvement in the separation time (roughly, 10-50 times) over the conventionally used slab-gel techniques. Moreover, the CZE method appears much more sensitive and amenable to component quantification and method automation.

Study of large DNA fragments in agarose gels by transient electric birefringence

The pulsed-field gel electrophoresis (PFG) is a newly developing technique used in the fractionation of large DNA fragments. Advances in PFG demand a better understanding in the corresponding mechanisms of DNA dynamics in the gel network. Detailed experiments are needed to verify and to extend existing theoretical predictions as well as to find optimum conditions for efficient separation of large DNA fragments. In the present study, deformation of large DNA fragments (40-70 kilobase pairs) imbedded in agarose gels were investigated by using the transient electric birefringence (TEB) technique under both singular polarity and bipolarity electric pulses at low applied electric field strengths (E less than or equal to 5 V/cm). The steady-state optical retardation (delta s) of DNA molecules is linearly proportional to E2. At a given E, the amplitude of optical retardation [delta(t)] increases monotonically with the pulse width (PW) and then reaches a plateau value [delta(t = 0) = delta s] where t = 0 denotes the time when the applied field is turned off or reversed. The field-free decay time (tau-a few minutes) is several orders of magnitudes slower than that from previous TEB observations using high electric field strengths (E-kV/cm) and short pulse widths (PW-ms). The degree of deformation (stretching and orientation) and the time of restoration to the equilibrium conformation of overall DNA chains have been related to delta and tau. In field inversion measurements, exponentially rising and linearly falling of birefringence signals in the presence of forward/inverse applied fields were observed. The rising and falling of birefringence signals were reproducible under a sequence of alternating pulses. Comparison of our results with literature findings and discussions with theories are presented.

Sizing of the Human T Cell Receptor a Locus and Detection of a Large Deletion in the Molt-4 Cell Line

The human T cell receptor alpha (TCR-alpha) chain gene consists of discontinuous DNA segments encoding multiple variable (V), multiple joining (J), and one constant (C) region. Unlike other immunoglobulin or TCR genes, however, the TCR-alpha locus exhibits an unusual dispersal of J segments that occupy a region of greater than 50 kilobases (kb) upstream of the C region, with the exact size still unknown. We report here the study of the TCR-alpha genetic locus by using pulsed-field gel electrophoresis (PFGE), which permits the separation of large DNA fragments. Analysis of DNA prepared from normal peripheral blood mononuclear cells, human endothelial cells, and a B cell line demonstrates that both V and C sequences are contained within a single 400-kb SfiI restriction fragment. PFGE analysis of the T cell line Molt 4 suggests a greater than 600-kb deletion involving the TCR-alpha gene.

Sizing of the human T cell receptor alpha locus and detection of a large deletion in the Molt-4 cell line

The human T cell receptor alpha (TCR-alpha) chain gene consists of discontinuous DNA segments encoding multiple variable (V), multiple joining (J), and one constant (C) region. Unlike other immunoglobulin or TCR genes, however, the TCR-alpha locus exhibits an unusual dispersal of J segments that occupy a region of greater than 50 kilobases (kb) upstream of the C region, with the exact size still unknown. We report here the study of the TCR-alpha genetic locus by using pulsed-field gel electrophoresis (PFGE), which permits the separation of large DNA fragments. Analysis of DNA prepared from normal peripheral blood mononuclear cells, human endothelial cells, and a B cell line demonstrates that both V and C sequences are contained within a single 400-kb SfiI restriction fragment. PFGE analysis of the T cell line Molt 4 suggests a greater than 600-kb deletion involving the TCR- alpha gene.