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.     

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.     

Microfluidic separation of DNA

DNA separation is important for numerous applications in biotechnology and medicine. Efforts to improve DNA separation in microdevices have led to advances in capillary electrophoresis and the development of novel separation strategies. Current research on microcapillary electrophoresis materials is focused on the development of separation matrices with low injection viscosities and wall-coating capabilities. Microcapillary injector geometries are being designed to allow increased control of sample plug volumes. Novel separation strategies using entropic traps, arrays of pillars and Brownian ratchets are also being developed.     

The impact of urea on viscosity of hydroxethyl cellulose and observed mobility of deoxyribonucleic acids

The influence of urea on the viscosity of hydroxyethyl cellulose (HEC), and the state and separation of double-stranded DNA, was studied by viscometry, fluorometry, and capillary electrophoresis. The results show that double logarithm plots of specific viscosity against the volume fraction of HEC in very dilute polymer solutions are linear, the slopes of which decrease from 0.96 in 0M to 0.29 in 7M urea. The linear regression plots converge at 0.0029 g/mL, the entanglement threshold of HEC. The inclusion of urea in HEC solution thus provides an accurate method of determining its entanglement threshold from such plots. Above the entanglement threshold of HEC, urea has no effect on the specific viscosity of HEC. Results also show that urea has no effect on double-stranded DNA. No change in fluorescence was observed when increasing amounts of urea were added to a fixed concentration of DNA. To examine the influence of urea on the migration of DNA in HEC, the separation of DNA was carried out by polymer-solution capillary electrophoresis in HEC solutions containing 0 or 7M urea using unmodified capillary. Observed mobilities were used in data reduction. It was found that a parallel relationship exists between the observed mobilities and the true mobilities. In buffers containing no urea, the pseudo-free solution mobility appears to be independent of the DNA size. It was also observed to be independent of the electric field below 300 V/cm, but relates exponentially to it in 7M urea. The pseudo-retardation constants obtained by Ferguson-like plots were observed to be positive for smaller DNA molecules below 300 V/cm and increasing linearly with electric field in 0M urea, but nearly constant in 7M urea.     

Poly(ethyleneoxide) for high resolution and high-speed separation of DNA by capillary electrophoresis

Capillary electrophoresis (CE) with polyacrylamide gels has already been demonstrated to allow single-base resolution of single-stranded DNA. However, linear polyacrylamide is not an ideal matrix because of a high viscosity and difficulties in preparing the polymer with well defined pore sizes. Alternatively, poly(ethyleneoxide) (PEO) with a large range of molecular masses from 300 000 to 8 000 000 is available commercially. In addition, it is easy to prepare homogeneous solutions to provide highly reproducible separation performance with sufficient resolution. Single-base resolution of double-stranded DNA between 123 and 124 base pairs can be achieved by the use of homogeneous matrices prepared from PEO (2.5% M_r 8 000 000), and even better resolution is achieved by using mixed polymer matrices. With further work, it should be possible to change the fractions and the total amounts of polymers to achieve even higher resolution for different samples with different size ranges of fragments. Another advantage of mixed polymer matrices is that relatively high resolution can be obtained while maintaining a relatively low viscosity compared to linear polyacrylamide with identical contents of formamide and urea, which makes it easier to fill these matrices into small capillaries.     

Capillary affinity gel electrophoresis

Capillary affinity gel electrophoresis is a new technique for the recognition of the specific DNA base and/or sequence. This technology is also applicable to the characterization of binding properties of DNA-based drugs, chiral separation, and the selective separation of antibody mimetics using imprinted polymers. This article reviews the present state of studies on the capillary affinity gel electrophoresis, including the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the detection of the mutation onDNA is illustrated.     

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.     

DNA analysis by microfabricated capillary electrophoresis device.

The LIGA (Lithographie Galvanoformung Abformung) process using synchrotron radiation lithography is applied to the microfabrication of capillary array electrophoresis (CAE) device. Laser-induced fluorescence detection system for the CAE device has been constructed by the modification of laser confocal fluorescence microscopy. DNA molecules were detected during migrating in the microchannels filled with polymer separation matrices under electric field to optimize the separation conditions for DNA analysis. Based on this observation, we demonstrated that microfabricated CAE device is realized the fast separation of DNA.     

Hydroxyethylcellulose as an effective polymer network for DNA analysis in uncoated glass microchips: optimization and application to mutation detection via heteroduplex analysis

The nature of the sieving matrix for DNA fragment separation is of immense importance in capillary and microchip electrophoresis. The chemical nature of the surface of the capillary or microchannel wall is equally as important, particularly with DNA electrophoresis where a substantial electroosmotic flow (EOF) may be detrimental to the separation. Although DNA analysis has been carried out successfully in both coated and uncoated capillaries, analysis of unpurified polymerase chain reaction products has been carried out primarily with covalently coated surfaces, especially with microchip electrophoresis. In this report, double-stranded (ds) DNA fragment analysis using hydroxyethylcellulose (HEC) buffered in 1xTris-borate-EDTA is demonstrated both in uncoated capillaries and in microchips. EOF was suppressed 20% in the presence of 1.5% HEC, and the effectiveness of HEC as a polymer for dsDNA fragment analysis was dependent on the pH, with pH 8.6 being optimal. Using separation efficiency (number of theoretical plates) and resolution to gauge the effectiveness of a variety of polymers for the capillary separation of dsDNA fragments in the size range 60-587bp, HEC was found to be comparable in performance to polydimethylacrylamide (PDMA), and superior to linear polyacrylamide and polyethylene oxide for DNA analysis. With respect to longevity and robust performance, HEC could be used effectively in an uncoated capillary for more than 40 runs and for more than 90 runs (without replenishing the polymer) in an uncoated microchip. Application of the optimized HEC conditions is demonstrated through its ability to facilitate heteroduplex analysis.     

Electrophoresis of long DNA molecules in linear polyacrylamide solutions

Electrophoresis of long DNA (T4 DNA; 166 kb, S. pombe chromosomal DNA; 3-6 Mb) in linear polyacrylamide solutions was investigated by fluorescence microscopy and capillary electrophoresis. In the past studies on electrophoresis of long DNA in a polymer solution, it was reported that DNA migrates in 'U-shape conformation'. We found that at higher polymer concentrations, the shape of the migrating DNA changes from U shape to linear shape ('I-shape conformation'). In the migration mode with the I-shape conformation, the DNA moves with almost constant velocity and constant shape. However, the migration velocity does depend on the DNA size, and it is possible to separate DNAs under this I-shape motion. Actually, Mb-sized DNAs are well separated within 5 min in the region for the I-shape motion by means of capillary electrophoresis with a DC field. Considering that it takes 20 h to separate Mb-sized DNAs by standard pulsed-field gel electrophoresis (PFGE), this results will be useful for the separation of giant DNAs.     

DNA sequencing by capillary electrophoresis (review)

DNA sequencing by capillary electrophoresis has been reviewed with an emphasis on progress during the last four years. The effects of sample purification, composition of sieving matrices, electric field strength, temperature, wall coating and DNA labeling on the DNA sequencing performance are discussed. Multicapillary array instrumentation is compared with one-capillary systems. Integrated systems that perform the whole DNA sequencing operation online starting from the DNA amplification through base calling and data processing are discussed.     

Determination of lysophosphatidic acids by capillary electrophoresis with indirect ultraviolet detection

Lysophosphatidic acid (LPA) is the simplest form of lysophospholipid. Molecular species of LPA have been identified as the potent components in the ovarian cancer activation factor. The elevated plasma LPAs may be used as potential biomarkers for the early detection of ovarian cancer. This paper is the first report on the quantitative analysis of molecular species of LPA using capillary electrophoresis. In this work, the separation of LPAs was achieved within 14 min in an adenosine monophosphate-borate–methanol–water solution, and the measurement was accomplished by indirect UV detection. With LPA (D) as internal standard, the method had linear calibration ranges for LPAs from 2.8 to 75 μM. The detection limits for various molecular species of LPA were from 1.2 to 2.3 μM by the pressure injection at 3.45 kPa for 5 s. The method had been applied to serum fortified with LPA (S), LPA (O), LPA (P), and LPA (M) and the recoveries ranged from 83 to 112%.     

无胶筛分毛细管电泳分离DNA片段的现状与展望

毛细管电泳已DNA片段分离分析的重要手段。本简述了毛细管电泳中采用无胶筛分介质分离DNA片段的机理研究,介绍了筛分介质近年的研究发展状况,依据分离介质的化学组成,分单聚物、共聚物和混聚物等3个部分进行了评述,并对其发展前景进行了展望。     

Purification and recovery of bulky hydrophobic DNA adducts.

For many years (32)P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of mass spectrometry in mind, the current research presents a new method to quantitatively purify bulky hydrophobic DNA adducts at levels that are pertinent to ongoing DNA adduct research in human health and environmental fields. This method was demonstrated with benzo[a]pyrene adducts. Purification was accomplished with the use of small columns (7.5-mm frits) with an 11 mg bed of polystyrene-divinlybenzene beads which retained the adducts while permitting the nonadducted nucleotides to be washed out with water. Subsequently, the adducts were eluted with 50% MeOH and the sample was reduced in volume in an evacuated centrifuge. Purification was demonstrated at adduct levels ranging from 4 adducts in 10(6) nonadducted nucleotides to 4 in 10(8). For these levels, analyses by capillary electrophoresis with sample stacking and UV detection determined that recoveries ranged from 91 to 54%, respectively. The adduct quantities isolated should be sufficient to allow the use of current MS capabilities that are linked on-line to separation methodologies such as capillary electrophoresis, capillary electrochromatography, and high-pressure liquid chromatography.     

Isotachophoresis of nucleic acids in agarose gel rods

A new method of electrophoresis (isotachophoresis in agarose gel rods) in which nucleic acid molecules are not separated but, oppositely, are brought together into one band, was elaborated. Heterogeneous in size DNA and RNA polymers present in a few milliliters of a solution at so low concentration that their isolation by other methods is hardly attainable and fraught with losses are brought together into one visible narrow band when put in a discontinuous electric field. Polynucleotides migrate in dilute (0.1%) semifluid agarose gel that permits easy quantitative isolation of the band of interest. Resulting DNA can be used directly in PCR. The suggested method for isolation of micro amounts of nucleic acids from dilute solutions can be applied to forensic and clinical research and cancer gene diagnostics by the analysis of fragmented circulating DNA from bodily fluids.     

Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis

We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction.     

Affinity capillary electrophoresis: important application areas and some recent developments

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting (`plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen–antibody, hapten–antibody, lectin–sugar, drug–protein, and enzyme–substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.     

Capillary affinity gel electrophoresis: new technique for specific recognition of DNA sequence and the mutation detection on DNA

The present state of studies on capillary affinity gel electrophoresis, which is a new technique for the specific recognition of a target DNA sequence, is reviewed. This article includes the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the sequence-specific recognition of DNA and the detection of mutations in specific genes is illustrated.     

Comblike, monodisperse polypeptoid drag-tags for DNA separations by end-labeled free-solution electrophoresis (ELFSE)

The development of innovative technologies designed to reduce the cost and increase the throughput of DNA separations continues to be important for large-scale sequencing and genotyping efforts. We report research aimed at the further development of a free-solution bioconjugate method of DNA size separation by capillary electrophoresis (CE), in particular, the determination of an optimal molecular architecture for polyamide-based "drag-tags". We synthesized several branched poly(N-methoxyethyl glycine)s (poly(NMEG)s, a class of polypeptoids) as novel friction-generating entities for end-on attachment to DNA molecules. A 30-mer poly(NMEG) "backbone," comprising five evenly spaced reactive epsilon-amino groups, was synthesized on solid phase, cleaved, and purified to monodispersity by RP-HPLC. Three different comblike derivatives of this backbone molecule were created by (1) acetylating the epsilon-amino groups or (2) appending small, monodisperse NMEG oligomers (a tetramer and an octamer). Grafting of the oligo(NMEG)s was done using solution-phase amide bond formation chemistry. Once purified to total monodispersity, the three different drag-tags were studied by free-solution electrophoresis to observe the effect of branching on their hydrodynamic drag or "alpha" and hence their ability to separate DNA. Drag was found to scale linearly with total molecular weight, regardless of branch length. The octamer-branched drag-tag-DNA conjugate was used to separate ssDNA products of 50, 75, 100, and 150 bases in length by free-solution CE in less than 10 min. Hence, the use of branched or comblike drag-tags is both a feasible and an effective way to achieve high frictional drag, allowing the high-resolution separation of relatively large DNA molecules by free-solution CE without the need to synthesize very long polymers.     

高速DNA序列分析

高速DNA序列分析是人类基因组研究的关键技术．文章对高速DNA序列分析方法如阵列毛细管电泳、超薄层凝胶板电泳、质谱、杂交法、原子探针法、流动单分子荧光检测法等新进展进行了评论．