Oligonucleotide analysis by gel capillary electrophoresis

Several million oligonucleotides are synthesized each year for a broad variety of molecular biology applications. Steady improvements in the synthesis chemistry efficiency and the automated DNA synthesizers have made production of oligonucleotides routine and reliable. Many applications, such as PCR and sequencing, are often successful when the primers have not been rigorously purified. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary for the dozens of oligonucleotides produced each day by a DNA synthesis laboratory. Traditional methods of analysis have been HPLC and polyacrylamide slab tel electrophoresis (PAGE). Gel capillary electrophoresis is a new option, combining the advantages of the HPLC and PAGE, with unprecedented resolution and speed.     

Three methods of capillary electrophoresis compared with high-resolution agarose gel electrophoresis for serum protein electrophoresis

We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins–Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins–Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus.     

High-pressure gel loader for capillary array electrophoresis microchannel plates.

Microfabricated capillary array electrophoresis (microCAE) microchannel plates are the next generation of bioanalytical separation devices. To fully exploit the capabilities of microCAE devices, supporting technology such as robotic sample loading, gel loading, microplate washing, and data analysis must be developed. Here, we describe a device for loading gel into radial capillary array electrophoresis microplates and for plate washing and drying. The microplates are locked into a loading module, and high-pressure helium is used to drive aqueous separation media or wash solutions into the microchannels through fixtures connected to the central anode reservoir. Microplates are rapidly (30 s to 5 min) loaded with separation media, such as 3%-4.8% linear polyacrylamide or 0.7%-3.0% hydroxyethyl cellulose, for electrophoresis. The effective and rapid gel-filling and plate-cleaning methods together with short electrophoretic analysis times (2-30 min) make microCAE systems versatile and powerful nucleic acid analysis platforms.     

Microchip capillary gel electrophoresis of multiply PEGylated high-molecular-mass glycoproteins

PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation ("MCGE fingerprinting") of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range.     

High-performance separation of polynucleotides by capillary gel electrophoresis.

Capillary gel electrophoresis was applied to the high speed separation of DNA and RNA. Factors affecting resolution and speed were optimized for the single base resolution of polynucleotides. Polynucleotides up to 350 bases were completely resolved within 38 min under optimum conditions.     

Microfabricated polymer injector for direct mass spectrometry coupling

This paper demonstrates the coupling of a plasma etched polymer microfluidic system with an electrospray mass spectrometer by generation of a nanospray. Taking advantage of the microtechnology processes and polymer properties, high volume production with good reproducibility of hydrophobic interfaces could be obtained. The nanospray was directly produced from the outlet of the plastic microfabricated chip positioned in front of the capillary entrance of the mass spectrometer. No chemical background due to the polymer has been observed under standard nanospray conditions. The performances of the spray as well as its efficiency have been demonstrated by flow measurements, stability establishment and tandem mass spectrometry experiment on angiotensin II. The spray was actuated without additional flow in methanol: water:acetic acid (50:49:1%) solution. A 40 fmol/microL detection limit could be reached.     

A micromachined capillary electrophoresis chip with fully integrated electrodes for separation and electrochemical detection

A novel analytical microsystem with fully integrated electrodes for electrophoresis and amperometrical detection is described. With respect to the lab-on-a-chip concept a capillary electrophoresis (CE) microsystem has been fabricated with a total of six gold electrodes for sample injection, separation and electrochemical detection using standard microfabrication technologies. The device is a ready-to-use system that does not need any extra mechanical apparatus for electrode insertion. The CE-chip has successfully been tested by measuring hydrogen peroxide, ascorbic acid and uric acid simultaneously. All three oxidizable species could be detected in less than 70 s. Glucose was detected by performing an enzymatic reaction along the separation channel. The microsystem showed a very good reproducibility.     

Single-based resolution for oligodeoxynucleotides and their phosphorothioate modifications by replaceable capillary gel electrophoresis

A replaceable capillary gel electrophoretic (replaceable CGE) method was developed for the separation of two sets of model compounds of single-stranded oligodeoxynucleotide mixtures (18-20 mers), phosphodiester oligodeoxynucleotides (PO-ODNs) and their phosphorothioate modifications (PS-ODNs), with equal sequences differing in a single base. Polyethylene glycol (PEG) 35000 was chosen as the sieving matrix. It was confirmed that PEG polymer solution less influenced resolutions of the PS-ODNs compared with those of the PO-ODNs, while acetonitrile used as an additive in the system improved the separation significantly. It was also noticed that the effect of temperature on separation was much larger than that of denaturant urea.     

Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis

Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.     

Steady-state gel electrophoresis of long polymer molecules: a theoretical study

The velocity of long polymer molecules in a gel and the liquid flow profile in the vicinity of a molecule's surface were studied theoretically by combining the Navier-Stokes equation with the Poisson-Boltzmann equation. The electrophoretic mobility has been calculated in dependence of the ionic strength of the electrolyte solution, its viscosity, the gels' volume friction coefficient, the surface charge and the radius of the polymer molecule. The results are presented in a non-dimensional form and depend on two dimensionless parameters only. The first parameter is the radius of the polymer molecule in units of the Debye length. The second is a parameter comprising the electrolyte's viscosity and the gel density. Thus, by similarity theory, the results apply to any given experimental arrangement. Received: 10 May 1999 / Revised version: 17 November 1999 / Accepted: 6 December 1999     

Electrohydrodynamic instabilities and segregation of polysaccharides in capillary polymer solution electrophoresis

During capillary electrophoresis of negatively charged polysaccharides in polymer solutions as sieving media, concentration fluctuations develop due to electrohydrodynamic instabilities caused by polarization of the polyelectrolytic chains. This leads to deviations from electroneutrality far beyond the Debye layer and segregation of the initially homogeneous sample solution into aggregated sample‐rich domains as verified by epifluorescence videomicroscopy imaging. As a result, anomalous and irregular peak profiles are obtained impeding the characterization of such complex sample mixtures. This effect appears at an electric field strength threshold value that depends on the molecular weight of the solute polymer molecules, pH, type and concentration of the polymer solution sieving media, and buffering conditions. The magnitude increases with increasing field strength and amount of sample injected. The aggregation onset, as evaluated by the value of the threshold potential, is affected by the charge density of the sample polymer molecules and Debye screening effects and investigated through variation of pH and ionic strength, respectively. Exchange of a simple base buffer component for small and multiply charged organic bases markedly increases the electric field strength necessary to trigger the electrohydrodynamic instabilities. Ultimately, the threshold value could be increased more than seven times by addition of an oppositely charged aminodextran polymer, thereby decreasing the analysis time. © 1999 John Wiley & Sons, Inc. Biopoly 49: 515–524, 1999     

Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis.

Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates. Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution. Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE). The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found. The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks. Additionally, the different isoforms can now be quantified by CGE. Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification. CGE also offers high resolution and the amount of DNA required is very low. Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation.     

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CV's below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.     

Quantitation of fluorescent nucleotide incorporation by capillary gel electrophoresis and laser-induced fluorescence detection

A method has been developed by which enzymatically incorporated fluorophore-labeled nucleotide sites in nucleic acid can be quantitated by degradation of nanogram quantities of DNA followed by capillary gel electrophoretic analysis with fluorescence detection. In this way the differing relative labeling densities achieved using either C5-substituted dUTP's or N4-substituted dCTP's were determined. The method has proven to be very useful in obtaining quantitative analytical data from the small quantities of complex molecules produced in nick translations. Various polymerization conditions using DNA polymerase I were examined to determine optimal labeling density. Simultaneous copolymerization of green fluorescing dCTP and dUTP nucleotides were undertaken in an attempt to maximize labeling density.     

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.     

Identification and quantification of GC-rich oligodeoxynucleotides in tissue extracts by capillary gel electrophoresis

Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50cm neutrally coated capillary at 60 degrees C together with a 7M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions >or=1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.