DNA sequencing by capillary electrophoresis

Capillary electrophoresis has been under development for DNA sequencing since 1990. This development has traveled down two parallel tracks. The first track studied the details of DNA separation by gel electrophoresis. Early work stressed rapid separations at high electric fields, which reached the extreme of a 3.5 min sequencing run at 1200 V/cm. While fast separations are useful in clinical resequencing applications for mutation detection, long read-length is important in genomic sequencing. Unfortunately, sequence read-length degrades as electric field and sequencing speed increases; this tradeoff between read-length and sequencing speed appears to be a fundamental result of the physics of DNA separations in a polymer. The longest sequence sequencing read-lengths have been obtained at modest electric fields, high temperature, and with low concentration noncrosslinked polymers. In parallel with our understanding of DNA separations, the second track of DNA sequencing development considered the design of large-scale capillary instruments, wherein hundreds of DNA samples can be sequenced in parallel. Real-world application of these very high throughput capillary electrophoresis systems will require significant investment in sample preparation technology.     

Characterization of DNA standards by capillary electrophoresis

We have used capillary electrophoresis to evaluate commercial DNA size standards and have found that it can provide an efficient assessment of size. However, the accuracy of the determination is adversely affected by anomalous migration times due to specific interactions of the DNA with the gel matrix as well as conformational differences in the DNA due to sequence heterogeneity. These anomalous migration times are strongly dependent on the choice of gel matrix. For example, the anomalous migration times that are observed in a 1 kilobase standard DNA ladder can be minimized using nongel hydroxyethylcellulose. In addition, the peak resolution can be increased and the anomalous migration can be reduced by the addition of the intercalating dye, ethidium bromide. However, in the case of the D1S80 allelic ladder, some of the DNA fragments possess nucleotide sequences which do not interact equivalently with the dye and produce irregular migration times. These measurements yield preliminary information useful in evaluating DNA size standards which may be used for a wide range of DNA diagnostic applications.     

Replaceable polymers in DNA sequencing by capillary electrophoresis

Much progress has been made in the development of replaceable sieving polymers and capillary coatings for high-performance DNA sequencing by capillary electrophoresis. Several studies of parameters that affect resolution, read length and reproducibility have begun to reveal the physical mechanisms acting on single-stranded DNA during electrophoresis through semidilute polymer solutions. Recently developed electro-osmosis-inhibiting matrix polymers have simplified the process of coating capillaries, facilitating the automation of high-throughput parallel systems for large-scale sequencing.     

Recent advances in capillary zone electrophoresis of DNA

The present minireview summarizes recent developments in the field of DNA separations by capillary zone electrophoresis (CZE), as developed by our group. Separation of antisense oligonucleotides in sieving liquid polymers in isoelectric buffers is discussed first. It is shown that the use of isoelectric buffers permits very high voltage gradients (up to 1000 V cm-1) with much reduced transit times and increased resolution of all truncated and failed sequences. Oligonucleotides can also be analysed by zone electrophoresis against a stationary pH gradient (typically a pH 6.5-10 range): if injected at the alkaline end, the sample components experience stacking and zone sharpening due to modulation of charge as the oligonucleotides move along the pH gradient. Oligonucleotides having the same length, but differing by one single nucleotide in the chain, can be separated in free solution (i.e. in the absence of a sieving matrix) at strongly acidic pH values (pH 3.0-3.3) where charge differences due to base protonation are maximized. By working in free solution, it has also been possible to measure accurately the free mobility of DNAs, shown to reach a constant value of 3.75 +/- 0.04 10(-4) cm2 V-1 s-1 at 25 degrees C and in Tris-acetate-EDTA buffer, pH 8.3, above a critical length of ca. 400 base pairs. Finally, detection of point mutations in human genomic DNA is proven to be feasible in nonisocratic CZE, by running temperature-programmed CZE. The temperature gradient is activated within the capillary lumen by voltage ramps during the run, by exploiting joule effects. This technique has been proven to work for all point mutants, from low-, to intermediate-, to high-melters and has been applied to a number of point mutants in cystic fibrosis and thalassemia.     

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.     

Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples

We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate and dideoxycytosine triphosphate; the concentrations of ddATP and ddCTP are adjusted to produce a 2:1 variation in the relative intensity of fragments. The second sequencing reaction uses a TAMRA labeled primer with dideoxythymidine triphosphate and dideoxyguanidine triphosphate; the concentrations of ddTTP and ddGTP are adjusted to produce a 2:1 variation in relative intensity of fragments. The pooled reaction products are separated by capillary gel electrophoresis and identified by one of three different detector systems. Use of a 2:1 peak height ratio typically produces a sequencing accuracy of 97.5% for the first 350 bases; a 3:1 peak height ratio improves accuracy to 99.5% for the first 400 bases. For these experiments, capillary electrophoresis is performed at an electric field of 200 V/cm; two to three hours are required to separate sequencing fragments up to 400 nucleotides in length.     

Analysis of antibiotics by capillary electrophoresis

The broad category of antibiotics encompasses some of the most widely prescribed pharmaceuticals in the world. As is the case with any pharmaceutical, an antibiotic must be characterized in terms of its potency or activity, and the presence and quantity of impurities. Additionally, any residue or metabolite that may be present as a result of its use must be monitored. Many capillary electrophoretic techniques have been utilized in the analysis of antibiotics, addressing the various aspects of their quantitation, profiling, and monitoring. Some of the more recent applications are summarized in this review article.     

Analysis of nucleotides by capillary electrophoresis

Capillary electrophoresis is a useful tool for the analysis of nucleotides. Methods have been optimized for both CZE and MECC modes. A variety of CZE buffers, such as borate, carbonate and phosphate were used successfully. The pH of the buffer changes the charge on the nucleotides. Therefore, the selectivity of the analytes can be controlled by the acidity of the buffer solution. CE separations of nucleotides have been performed at all pH levels, in both CZE and MECC modes. SDS was the most commonly used modifier in MECC separations, but other additives have been added to optimize selectivity. In addition, nucleotides have been quantified in different matrices, including tissue and cell extracts and several DNA and RNA sources. This paper summarizes the methods used for the optimization of nucleotides by CE and includes the most recent techniques to improve selectivity, reproducibility and sensitivity. A summary of CE methods is used in analyses of nucleotides in biological matrices is included.     

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits ( < 10 micrograms/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.     

Urine protein analysis by capillary electrophoresis

Increased concentration of proteins in urine as well as abnormal patterns are seen in many disorders such as various renal disorders and light chain disease. At the wavelength of 214 nm used for detection of the peptide bond, numerous compounds interfere in the analysis of urinary proteins. We show that either adsorptive filtration with a wash step or cold ethanol precipitation are two methods which can eliminate many of the interferences. The wash step is rapid, greatly reduces the interfering substances, and decreases the effect of sample matrix. Both of these methods yield results comparable to the traditional agarose method. Capillary electrophoresis (CE) is faster and more cost-effective than agarose electrophoresis.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

In a previous study an apparent discrepancy was found between the radiobiological hypoxic fraction of tumours and the tumour oxygenation: the lowest percentage of low pO2 values was observed in the most hypoxic tumour, a heavily pigmented melanoma Na11+. This report describes a similar study with two other less pigmented melanomas. The influence of melanin on pO2 readings was also studied using synthetic melanin and L-tyrosine. Tumour oxygenation was measured using the KIMOC 6650 histograph, apparent pO2 was also measured in the calibration chamber in a buffer containing melanin or L-Tyr at three pHs (6.5, 7.0, 7.5) and bubbled with three different oxygen concentrations (0.2, 2.0, 20.9%). The proportion of hypoxic cells, measured by an in vivo/in vitro colony assay, was 58% for Na11+, 30% for Be11 and 51% for Ma11 tumours. The melanin content (microgram/10(6) cells) was 6.5 (Na11+), 2.0 (Be11), and 4.3 (Ma11). The percentages of radiobiologically hypoxic cells and low pO2 reading values (<2 mmHg) were inversely correlated, contrary to what was expected. In buffer, the pO2 values increased significantly with the melanin concentration: the lower the oxygen concentration, the greater was the increase in pO2. The pO2 readings values increased to a lesser extent with L-Tyr concentration. These results indicate that clinical determination of pO2 in melanoma tumours requires careful attention.     

Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.     

Detection of 2'-deoxyoxanosine by capillary electrophoresis

We have investigated capillary electrophoresis separation of oxanine (Oxa), a base moiety of 2'-deoxyoxanosine (Figure 1), from various bases. Oxa was not well separated from the others by micelle electrokinetic chromatography which is a general method for separating of the bases. On the other hand, capillary zone electrophoresis showed much improved separation when used pH 12 where the six-membered ring of Oxa is opened.     

Serum protein electrophoresis by CZE 2000 clinical capillary electrophoresis system

We compared the automated Paragon 2000 clinical capillary zone electrophoresis (CZE) system with two manual methods, agarose electrophoresis (AGE) and cellulose acetate electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the alpha1-globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (alpha1-globulin). Total precision was between 0.8% (albumin) and 5.3% (beta-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.     

Separation of chiral compounds by capillary electrophoresis

This review presents the different chiral selectors used in capillary electrophoresis (CE) for the separation of enantiomers. The use of charged cyclodextrins, crown ethers, polysaccharides, proteins, natural and synthetic micelles, macrocyclic antibiotics and ergot alkaloids is discussed in detail. Neutral native and derivatized cyclodextrins are not treated because several review articles have already been published on this topic. Recent developments like the application of two chiral selectors in the same background electrolyte are highlighted.     

Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders

AIM: To facilitate DNA mutation analysis by use of capillary electrophoresis. METHODS: The usefulness and applications of capillary electrophoresis in DNA fragment sizing and sequencing were evaluated. RESULTS: DNA mutation testing in disorders such as cystic fibrosis, Huntington disease, alpha thalassaemia, and hereditary fructose intolerance were undertaken effectively. However, sizing the (CAG)n repeat in the case of Huntington disease was a potential problem when using capillary electrophoresis. Separation polymers used in capillary electrophoresis are still in the developmental phase, with improved ones being released regularly. CONCLUSIONS: In the DNA diagnostic setting, capillary electrophoresis is a valuable development because it expands the scope for automation and has useful analytical properties. The potential to perform complex multiplexing within one electrophoresis run facilitates DNA diagnosis. The different mobility of DNA fragments in capillary electrophoresis compared with conventional gel electrophoresis will require, in some circumstances, additional care when results are being interpreted or reported. Capillary electrophoresis is a cheap alternative for combined automated sequencing and fragment analysis that utilises multicolour fluorescence capability. However, in its present form, it is not useful for large scale sequencing.     

Profiling of oligosaccharide-mediated microheterogeneity of a monoclonal antibody by capillary electrophoresis

Pro-haemolysin (approximately 110 kDa), the inactive precursor of the membrane-lytic toxin alpha-haemolysin, has been purified from an overproducing strain of Escherichia coli. Pro-haemolysin forms aggregates in aqueous media, like the mature protein, suggesting an amphipathic structure. Direct measurements of protein binding to liposomal membranes, following a novel procedure, show that pro-haemolysin can bind the lipid bilayers to a similar extent as alpha-haemolysin. This is confirmed by the observed changes in the intrinsic fluorescence emission of the protein upon binding the bilayers. However, pro-haemolysin is totally unable to induce liposomal membrane lysis. Binding of Ca2+, that is essential for the lytic activity of alpha-haemolysin, is greatly diminished in the precursor protein, as shown both by direct measurements of 45Ca(2+)-binding and by fluorescence measurements. The results suggest that binding of a fatty acyl residue in the activation step brings about an important conformational change in the protein that involves the Ca(2+)-binding domain.     

Profiling glycoprotein n-linked oligosaccharide by capillary electrophoresis

A method for analysis of N-linked oligosaccharides derived from glycoproteins including sialic acid-containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. Glycoproteins were heat-denatured in the presence of a reducing agent and the N-linked oligosaccharides were released by peptide N-glycosidase (PNGase F; EC3.5.1.52)-catalyzed hydrolysis. The released N-linked oligosaccharides were derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N-linked oligosaccharide, desialylated, galactosylated biantennary, core-substituted with fucose (A2F) was tested for APTS-based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N-linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resolution of the N-linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.     

Exoglycosidase matrix-mediated sequencing of a complex glycan pool by capillary electrophoresis

This paper discusses oligosaccharide sequencing by consecutive enzymatic digestion of carbohydrates using an exoglycosidase array, followed by capillary electrophoresis separation of the digests. Because of the high resolving power and good reproducibility of capillary electrophoresis, multistructure sequencing of a complex glycan pool can be performed in most instances requiring no prior isolation of the individual oligosaccharides. High sensitivity laser-induced fluorescence detection enables acquisition of complete sequence information from several picomoles of glycoproteins. Comparison of the migration times of the exoglycosidase digest fragments to the maltooligosaccharide ladder, enables calculation of migration shifts, due to cleavage based on the actual exoglycosidases used. The particular sequence of each oligosaccharide in a glycan pool can be proposed with high confidence based on the migration time shifts of the various oligosaccharide structures. However, possible combinations of various sequence fragments may have very similar charge to hydrodynamic volume ratios, resulting in electrophoretic co-migration when a mixture of different oligosaccharides is sequenced together. Then, capillary electrophoresis separations of the resulting fragments should be evaluated after each digestion step. In the instances of complex separation profiles when multiple peaks are present, the evaluation of peak shifts can get very complicated and solved only with the aid of a software program. Data about the monosaccharide composition of the glycan pool provides useful information in designing the digestion enzyme matrix.     

Purity testing of recombinant proteins by capillary electrophoresis

Purity testing of recombinant DNA (rDNA) proteins using slab gel electrophoresis in conjunction with scanning densitometry is time consuming and labor intensive and is difficult to reproduce because the dyes used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes a micellar capillary zone electrophoresis (MCZE) procedure that overcomes these difficulties. The MCZE method was evaluated to estimate protein purity of hydrophobic cytomegalovirus proteins, expressed E. coli, and highly glycosylated hepatitis C virus proteins, expressed in Chinese hamster ovary cells. The results obtained by the MCZE procedure correlated very well with the purity results quantitated by the conventional slab gel electrophoresis method using purified Coomassie Brilliant Blue dye to reduce anomalies. MCZE may serve as an alternative method for in-process and purity testing of rDNA proteins.