Pulsed Field Capillary Electrophoresis Research Articles

Feasibility of Optical Genome Mapping from Placental and Umbilical Cord Sampled after Spontaneous or Therapeutic Pregnancy Termination

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Separation of proteins by square-wave pulsed field and inversion field capillary electrophoresis

BackgroundCapillary electrophoresis (CE) is an effective method for protein analysis. Although previous researchers have proved that the separation performance of large DNA molecules was improved by pulsed field capillary electrophoresis (PFCE), there is lack of research about proteins separation by PFCE. MethodsHerein, we systematically investigated the separation performance of proteins sized from 44 kDa to 200 kDa under square wave and inversion electric field, respectively. FindingsResults showed that for pulsed electric field with same forward and backward time duration, there was a resonance frequency for protein molecule re-orientation and applied frequency, and the apparent mobility (μ) was the lowest at the resonance frequency. μ was positively correlated to modulation depth (M) if M was below 200%, while μ was negatively correlated to M if M was higher than 200%. For CE with same forward and backward voltage, it demonstrated that μ dropped as M went up, but the separation performance for proteins with high molecular mass improved. Through PFCE, albumin and α- globulins in human serum were also resolved within 7 min. Such a work may shed new light on the analysis of proteins by PFCE.

Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10.

Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.

Is pulsed electric field still effective for RNA separation in capillary electrophoresis?

Pulsed field capillary electrophoresis (PFCE) is a predominant technique to cope with difficulties in resolving large DNA strands, yet it is still unclear whether pulsed electric field is effective for the separation of higher mass RNA. In this paper we focused on the role of pulsed electric field in large RNA fragments analysis by comparing RNA separation performance in PFCE with that in constant field CE. Separation performance in terms of migration mobility, plate numbers, resolution, and selectivity has been tested for the analysis of RNA from 0.1 to 10.0 kilo nucleotide (knt) under different electrophoretic conditions. Denaturation, important to obtain uniform and identifiable peaks, was accomplished by heating the sample in 4.0M urea prior to analysis and the presence of 4.0M urea in the electrophoresis buffer. Results demonstrate that unlike DNA in PFCE, the pulsed electric field mainly affects the separation performance of RNA between 0.4 and 2.0knt. The migration mobility of long RNA fragments is not a strong function of modulation depth and pulsed frequency. Moreover, the logarithm of RNA mobility is almost inversely proportional to the logarithm of molecule size up to 6.0knt with correlation coefficient higher than 0.99 in all the polymer concentrations measured here. Resonance frequency of RNA in PFCE was also observed. While these initial experiments show no distinct advantages of using PFCE for RNA separation, they do take further step toward characterizing the migration behavior of RNA under pulsed field conditions.

Separation of long DNA fragments by inversion field capillary electrophoresis

This study reports improved pulsed field capillary electrophoresis (PFCE) for separation of large DNA ladders. Important analytical conditions, including gel polymer concentration, ratio of forward to backward pulse duration, and separation potential, were investigated for their effects on the separation performance of DNA ranging in size from 0.1 to 10.0 kilo base pairs (kbp). Results show that DNA fragments from 0.1 to 8.0 kbp can be resolved with high resolution, simultaneously, in a short time. The ratio of forward to backward pulse duration affects the separation performance for DNA fragments greater than 1.5 kbp, and 3 or 4 is the optimum value of the ratio for separation of DNA up to 10 kbp. Furthermore, the separations that were obtained with 74-19,329 bp λ-DNA restriction fragments clearly demonstrate a dramatic improvement in the separation time and resolution over the conventionally used square-wave PFCE. The inversion field capillary electrophoresis reported here may help enable future DNA analysis studies to be performed quickly and effectively.

Acetic acid denaturing pulsed field capillary electrophoresis for RNA separation

Based on our previous work of in-capillary denaturing polymer electrophoresis, we present a study of RNA molecular separation up to 6.0 kilo nucleotide by pulsed field CE. This is the first systematic investigation of electrophoresis of a larger molecular mass RNA in linear hydroxyethylcellulose (HEC) under pulsed field conditions. The parameters that may influence the separation performance, e.g. gel polymer concentration, modulation depth and pulse frequency, are analyzed in terms of resolution and mobility. For denaturing and separating RNA in the capillary simultaneously, 2 M acetic acid was added into the HEC polymer to serve as separation buffer. Result shows that (i) in pulsed field conditions, RNA separation can be achieved in a wide range of concentration of HEC polymer, and RNA fragments between 0.3 and 0.6 kilo nucleotide are sensitive to the polymer concentration; (ii) under certain pulsed field conditions, RNA fragments move linearly as the modulation depth increases; (iii) 12.5 Hz is the resonance frequency for RNA reorientation time and applied frequency.

Effect of temperature on the separation of long DNA fragments in polymer solution

Electrophoresis of long DNA fragments in polymer solutions is still attractive when performed in short capillaries. Then the separations can be accomplished in minutes rather than hours as is usual in various slab electrophoresis techniques. In this paper we focused on the behavior of large DNA fragments in pulsed field capillary electrophoresis under various temperature conditions. The mobility dependence of fragments of λ-DNA single-cut mixture on various frequencies at three different temperatures showed that the antiresonance mobility minima are shifted to higher frequencies at higher temperatures. This interesting result is explained in terms of the geometration model of DNA motion.

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 ().

Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA.

We present a study aimed at understanding the factors affecting the separation of large DNA molecules by capillary electrophoresis in polymer solutions. In a first series of experiments, a systematic study of the effect of operational parameters on the development of an electrohydrodynamic instability resulting in DNA aggregation and spurious peaks in the electropherograms is presented. The results are discussed in regard to a recent theory of electrohydrodynamic instabilities in macroion suspensions, recently proposed by Isambert et al. Overall, the results provide strong support to the theory. Some situations of interest for applications, and not explicitly considered in the theory, such as asymmetric field pulsing and the use of polymer additives in the buffer, were also considered. Furthermore, robust optimized protocols for high resolution separation of DNA in the range of 100 base pairs to 160 kilobase pairs, are proposed. As predicted by the model, it is shown that using a concentrated isoelectric buffer (histidine) strongly reduces aggregation as compared to the use of a conventional buffer at the same concentration, and allows separation of DNA from 100 bp to 160 kbp in less than 10 min. We also present a systematic study of the dependence of the mobility vs DNA size, pulse frequency, and field strength. The results are discussed with respect to the Biased Reptation with Fluctuations model and a good agreement is obtained. Copyright 1999 John Wiley & Sons, Inc.

DNA sequencing with pulsed-field capillary electrophoresis in poly(ethylene oxide) matrix.

DNA sequencing by using pulsed-field capillary electrophoresis in a linear polymer matrix was performed in this work. The effects of waveform, frequency, modulation depth and heating were investigated. The separation performance for Sanger DNA fragments up to 1000 bp using a pulsed field was compared to isoelectric and isorheic conditions. A separation mechanism in the flexible poly(ethylene oxide) (PEO) gel was also suggested. We found that temperature change generated by either a pulsed field or ultrasound during DNA sequencing can have a deleterious effect on the separation efficiency.

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.

Rapid pulsed field capillary electrophoretic separation of megabase nucleic acids.

Pulsed field capillary electrophoresis in ultradilute solutions of sieving polymers has been used to separate nucleic acid fragments as long as 1.6 million base pairs. Hydroxyethyl cellulose solutions are used for separations in the size range 8000-50,000 base pairs. Longer chain nucleic acids are separated in mixed hydroxyethyl cellulose/poly(ethylene oxide) solutions. Separations are rapid (12-13 min).

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.

Pulsed field capillary electrophoresis of multikilobase length nucleic acids in dilute methyl cellulose solutions.

Pulsed field capillary gel electrophoresis in dilute methyl cellulose solutions is used to separate nucleic acid fragments in the size range 75-23,000 base pairs. Field inversion is shown to increase resolution for fragments longer than about 500 base pairs. Methyl cellulose solutions as dilute as 0.01% can be used. Intermolecular hydrogen bonding is suggested as the cause of apparent cellulose fiber entanglement at concentrations below the calculated entanglement limit. The 1-kb DNA ladder and the lambda DNA/HindIII restriction fragment mixtures are each baseline resolved in a 28-cm capillary in less than 9 min at 180 V/cm (dc component).

Pulsed-field capillary electrophoresis: optimizing separation parameters with model mixtures of sulfonated polystyrenes.

The electrophoretic transport of high molecular weight charged solutes, both flexible and stiff polymers, has been studied by capillary electrophoresis under constant-field and pulsed-field conditions. Sulfonated polystyrenes were used as model solutes in different entangled polymer solutions. First, changes of the end-to-end distance vectors of flexible polymers were examined through the mobility/potential-gradient curves. Under pulsed-field conditions, the influence of different pulse shapes, frequencies, and amplitudes of forward and backward pulses on the electrophoretic mobilities of model solutes was studied. Resolution of the mixture components was strongly affected by changes in frequency of both sine-wave and square-wave pulses. The experimental results obtained under pulse-field conditions are roughly in agreement with the existing theories of electrophoretic transport.

Wave form fidelity in pulsed-field capillary electrophoresis

Depending on the conditions of capillary operation, application of a pulsed electric field was found not to yield the expected wave shape throughout the column. This distorsion was a result of the significant RC time constant formed from the very high solution resistance and the capacitance of the capillary.