Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments.

Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.     

Anomalous electrophoretic mobility of mouse mtDNA restriction fragments

Analysis of the electrophoretic mobilities of mouse mtDNA restriction fragments revealed a high incidence of anomalous migration in polyacrylamide slab gels. Relative to the mobility predicted by the sequence, 6 of 29 200- to 700-bp fragments had deviations of 5-12%. Three of these fragments migrated more slowly than predicted while three were faster. There was little, if any, correlation between electrophoretic mobility and base composition. The anomalous restriction fragments mapped throughout the mitochondrial genome.     

Unusual capillary electrophoretic behavior of triplet repeat DNA

We investigated the electrophoretic behavior of triplet repeat DNA fragments by capillary electrophoresis and found triplet repeat DNA fragments showed unusual mobilities compared with those of commercially available DNA molecular marker. The electrophoretic data are analyzed by means of Ogston model and the mechanism of a change in mobility of triplet repeat DNA is discussed. The unusual mobilities are caused by the characteristic higher-order structure formed by GC-rich triplet repeat DNA.     

Electrophoresis of DNA in ultrathin planar-format linear polyacrylamide

Linear or un-cross-linked polyacrylamides have been employed successfully in the field of capillary electrophoresis for the separation of nucleic acids. Typical acrylamide concentrations for those applications range from 3% to 14% (wt/vol), with consistencies ranging from virtually liquid to moderately viscous. Due to the absence of cross-links, and the relatively fluid nature of linear polyacrylamide at typically employed concentrations, its use in planar (slab) gel electrophoresis has been overlooked. We describe herein the application of ultrathin (100 μm) high-viscosity slabs of linear polyacrylamide to planar electrophoresis of nucleic acid fragments. The approach we describe is rapid and yields high-resolution separations of nucleic acid fragments in linear polyacrylamide supports. The mobilities of DNA fragments of various lengths in a range of concentrations of linear polymer are compared with those observed for conventional cross-linked gels. The reptative migration of larger DNA fragments in linear polymers is predictable from the models derived from work with cross-linked acrylamide and agarose. The migration of smaller fragments, however, is not entirely predicted by the Ogston model. The relative mobilities observed for very small DNA fragments are approximately half those predicted by the Ogston regimen.     

Single-stranded DNA fragments holding unusual conformation

In the chemical synthesis of DNA, we found that the single-stranded DNA (ssDNA) fragments containing the sequence GCGAAAGC showed higher mobilities than the fragments without this sequence on a denaturing polyacrylamide gel electrophoresis. Physical structure of these DNA fragments was studied by enzyme digestion and optical analysis. The abnormal mobilities on electrophoresis seem to depend on an unusual conformation.     

Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis

Capillary electrophoresis (CE) with a sieving buffer containing ethidium bromide was applied to the detection of PCR-amplified RFLP samples. With CE, in contrast to agarose gel electrophoresis, run times are short, i.e., typically less than 30 min, the capillary can be re-used, and full automation is feasible. The addition of ethidium bromide to the buffer system in conjunction with a field amplification injection technique led to increased sample detectability and resolution. Migration time precision was better than 0.2% RSD with a approximately 12-bp resolution for the DNA fragment sizes of interest. RFLP samples were analyzed for homo- or heterozygosity based on the presence of 500- and/or 520-bp DNA fragments. Special software was used to correct for run-to-run migration time variations, thus facilitating genotype assignment.     

Experimental and theoretical studies of DNA separations by capillary electrophoresis in entangled polymer solutions.

Studies of electrophoretic separations of DNA restriction fragments by capillary electrophoresis in solutions of (hydroxyethyl) cellulose (HEC) were performed. Rheological studies were used to confirm that the entanglement threshold (phi*) for the HEC solutions used is approximately 0.004 g/mL, in good agreement with theoretical predictions. A mesh size an order of magnitude smaller than that found in agarose gels (on a per weight basis) was calculated using polymer-entanglement theory and was confirmed by electrophoretic measurements. Electrophoretic migration was shown to follow the Ogston regime under most conditions. An approach for obtaining smaller mesh sizes is presented.     

Electrophoretic Mobility of DNA in Solutions of High Ionic Strength

The free-solution mobilities of small single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) have been measured by capillary electrophoresis in solutions containing 0.01–1.0 M sodium acetate. The mobility of dsDNA is greater than that of ssDNA at all ionic strengths because of the greater charge density of dsDNA. The mobilities of both ssDNA and dsDNA decrease with increasing ionic strength until approaching plateau values at ionic strengths greater than ∼0.6 M. Hence, ssDNA and dsDNA appear to interact in a similar manner with the ions in the background electrolyte. For dsDNA, the mobilities predicted by the Manning electrophoresis equation are reasonably close to the observed mobilities, using no adjustable parameters, if the average distance between phosphate residues (the b parameter) is taken to be 1.7 Å. For ssDNA, the predicted mobilities are close to the observed mobilities at ionic strengths ≤0.01 M if the b-value is taken to be 4.1 Å. The predicted and observed mobilities diverge strongly at higher ionic strengths unless the b-value is reduced significantly. The results suggest that ssDNA strands exist as an ensemble of relatively compact conformations at high ionic strengths, with b-values corresponding to the relatively short phosphate-phosphate distances through space.     

Typing dinucleotide repeats under nondenaturing conditions with single-base resolution and high sizing precision

Dinucleotide repeats are genetic markers that are useful for many purposes, including genetic epidemiology, population genetics, and genetic diagnostics. The accuracy of analyses based on dinucleotide repeat polymorphisms is highly dependent on the success achieved in minimizing genotyping errors. Genotyping errors in dinucleotide repeat typing may arise for various reasons, including polymerase chain reaction (PCR) processing errors and the use of unsuitable electrophoretic conditions for resolving amplification products (i.e., lack of single-base resolution and inadequate precision in allele sizing). We have recently described a nondenaturing electrophoretic system useful for detecting PCR processing errors that lead to misidentification of heterozygotes as homozygotes in (AC)_n repeat typing. Here, we show that this system also allows resolution of (AC)_n repeats in native conditions with single-base resolution and high sizing precision, on the basis of an analysis of seven human (AC)_n repeats ranging in size from 72 to 217 bp. This PAGE system is thus also useful for reducing the likelihood both of allele misidentification due to the absence of single-base resolution and of inaccuracies in allele sizing due to anomalous electrophoretic migrations among the alleles within an (AC)_n repeat.     

Towards a quantitative free flow electrophoresis and its application to particle size separations

The possibility to quantify free-flow electrophoresis (FFE) data was explored in application to 6 negatively charged polystyrene size standards in the size range of 73 to 762 nm diameter. Peak fraction numbers in FFE were shown to be proportional to mobilities of the particles, determined by capillary zone electrophoresis in the identical buffer. Standard deviations of peak fraction numbers demonstrate a high degree of intra-experimental reproducibility while inter-experimentally, a variability of 1 to 5 peak fraction numbers within 28 fractions was found. A relative mobility (Rf) scale for peak identification in FFE based on the free mobility of the dye, SPADNS, allowed for the utilization of the entire electrophoretic migration path but failed to improve the precision of fraction numbers in view of the substantial zone spreading of the dye. Mobility differences between particles increased upon lowering the ionic strength of the electrophoretic buffer. Peak width increased with particle size in inverse relation to ionic strength.     

Usefulness of microchip electrophoresis for reliable analyses of nonstandard DNA samples and subsequent on-chip enzymatic digestion.

The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-1), error increased in a dose-dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.     

Capillary electrophoresis of DNA in uncrosslinked polymer solutions: evidence for a new mechanism of DNA separation

The electrophoretic separation of DNA molecules is usually performed in thin slabs of agarose or polyacrylamide gel. However, DNA separations can be achieved more rapidly and efficiently within a microbore fused silica capillary filled with an uncrosslinked polymer solution. An early assumption was that the mechanism of DNA separation in polymer solution(SINGLEBOND)capillary electrophoresis (PS(SINGLEBOND)CE) is the same as that postulated to occur in slab gel electrophoresis, i.e., that entangled polymer chains form a network of "pores" through which the DNA migrates. However, we have demonstrated that large DNA restriction fragments (2.0(SINGLEBOND)23.1 kbp) can be separated by CE in extremely dilute polymer solutions, which contain as little as 6 parts per million [0.0006% (w/w)] of uncrosslinked hydroxyethyl cellulose (HEC) polymers. In such extremely dilute HEC solutions, far below the measured polymer entanglement threshold concentration, pore-based models of DNA electrophoresis do not apply. We propose a transient entanglement coupling mechanism for the electrophoretic separation of DNA in uncrosslinked polymer solutions, which is based on physical polymer/DNA interactions. (c) 1996 John Wiley & Sons, Inc.     

Oligo[d(C).(G)] runs exhibit a helical repeat of 11.1 bp in solution and cause slight DNA curvature when properly phased.

We have inserted d(C)10 in a set of DNA fragments with bent segments on both ends, which are rotated with respect to each other by base pair wise increasing insertions. The electrophoretic mobilities on polyacrylamide gels of these DNA fragments were used to identify insertion sizes with cis conformations of the bent ends. These experiments revealed a helical repeat in solution of d(C).d(G) tracts of 11.1 +/- 0.08 bp. The electrophoretic mobilities of ligation ladders with properly phased d(C)5 and d(C)16 runs demonstrate a small but clearly detectable curvature of these fragments.     

A computer program for determining the size of DNA restriction fragments

A computer program has been developed for determining the sizes of DNA restriction fragments from their electrophoretic mobilities. A parabola is fitted to the mobilities of a set of standard fragments of known sizes and the sizes of the unknown fragments are then calculated from the fitted curve. This procedure is shown to yield estimated sizes which are accurate to within a few percentage, as judged by experiments with fragments obtained by digestion of pBR322 with the restriction endonuclease HaeIII. The program, which is written in BASIC, is simple to use and is very much faster than the graphical method that it replaces.     

Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol

A simple and sensitive capillary zone electrophoresis method with UV absorbance detection is described for the quantitation of allopurinol and its metabolite oxypurinol in aqueous solution. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was systematically investigated; these parameters included the nature and concentration of the separation buffer, pH and applied voltage. A buffer consisting of 15 mM 2-[N-cyclohexylamino]ethanesulfonic acid (CHES) adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of these compounds. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing these compounds in serum and allopurinol in commercial pharmaceutical preparations.     

Separation of DNA fragments and single strand conformation polymorphism analysis in bare capillaries using poly(acrylamide–dimethylacrylamide) as a separation medium

A short chain poly(acrylamide–dimethylacrylamide) (PADMA) was synthesized in aqueous phase using isopropanol as a chain transfer agent, and was characterized according to the chemical composition and molecular mass. This polymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA–capillary wall interaction. The sieving medium has low viscosity and capillary filling with this medium and medium replacement were conveniently carried out by commercial capillary electrophoresis instruments. The effects of components and concentration of copolymers on the separation of DNA fragments were investigated. Highly efficient separation of DNA fragments, successful single strand conformation polymorphism (SSCP) analysis and good reproducibility of the migration time were obtained in bare capillaries using these copolymers as sieving media. Our preliminary results demonstrate that PADMA will become an alternative matrix for DNA separation by capillary electrophoresis.     

Bacteria genome fingerprinting by flow cytometry.

BACKGROUND: A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus in this study. METHODS: The isolation of the bacterial genomic DNA and the subsequent complete digestion by a restriction endonuclease were performed inside an agarose plug. Electroelution was used to move the DNA fragments out-of the agarose plug into a solution containing low concentrations of spermine and spermidine, added to stabilize the large DNA fragments. DNA was stained with the bisintercalating dye thiazole orange homodimer (TOTO-1) and subsequently introduced into our ultrasensitive flow cytometer from a capillary. RESULTS: Individual DNA fragments up to 351 kbp were successfully handled and sized. The histograms of the burst sizes were generated from signals associated with individual fragments in <7 min with <2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes approximately 20 h and requires approximately 1 microg of DNA with a sizing accuracy of approximately 90%. CONCLUSIONS: With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.     

A normalization strategy applied to HiCEP (an AFLP-based expression profiling) analysis: Toward the strict alignment of valid fragments across electrophoretic patterns

Background  

Gene expression analysis based on comparison of electrophoretic patterns is strongly dependent on the accuracy of DNA fragment sizing. The current normalization strategy based on molecular weight markers has limited accuracy because marker peaks are often masked by intense peaks nearby. Cumulative errors in fragment lengths cause problems in the alignment of same-length fragments across different electropherograms, especially for small fragments (< 100 bp). For accurate comparison of electrophoretic patterns, further inspection and normalization of electrophoretic data after fragment sizing by conventional strategies is needed.     

Analysis of polypeptide molecular weights by electrophoresis in urea

Ten proteins of differing disulfide contents and isoionic points were subjected to disc gel electrophoresis in the presence of 8 urea-0.9 acetic acid to evaluate the use of this technique in determining polypeptide molecular weights. Comparison of the electrophoretic mobilities before and after reduction of the proteins' disulfide bonds demonstrated that only after all disulfide bonds were broken, could their molecular weights be estimated with any degree of accuracy. The expression of the electrophoretic mobilities as a function of the proteins' effective hydrodynamic sizes, thereby taking into account the extent of constraint by disulfide bonds, allowed a comparison of disulfide cross-linked and linear forms of the protein polypeptides. The extent to which intrinsic charge affects a protein's electrophoretic mobility was estimated by comparing alpha-lactalbumin and lysozyme, two proteins of identical size but vastly different isoionic points. They exhibited a 20% difference in mobilities. An apparent slow reduction of disulfide bonds was observed to occur when proteins were exposed to reducing agent at low pH in 8 urea.