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.     

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.     

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.     

Two-dimensional separations: capillary electrophoresis coupled to channel gel electrophoresis

Two-dimensional separations provide extremely high peak capacities. Coupling capillary zone electrophoresis with ultrathin channel gel electrophoresis offers a convenient and efficient way to perform such two-dimensional microseparations. By means of in situ polymerization, high-concentration (up to 50%T) polyacrylamide gels are prepared in 75 mm long, 25 mm wide, and 40 microns thick rectangular channels. By moving the outlet end of the capillary electrophoresis capillary across the entrance of the channel, both separations are completely preserved. Mixtures of peptides labeled by fluorescein isothiocyanate (FITC) are well resolved in less than 15 min, with theoretical plate numbers in the range of 20,000-50,000 for each independent separation. Significant enhancement in separation efficiency and peak capacity over one-dimensional separations are demonstrated by this combination. The two-dimensional separations of a model mixture of peptides, a tryptic digest of trypsinogen, and < 0.05% of an individual B2 neuron from the marine mollusk Aplysia californica are presented.     

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.     

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.     

Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.     

Rapid DNA sequencing of more than 1000 bases per run by capillary electrophoresis using replaceable linear polyacrylamide solutions

The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.     

Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing

An integrated and multiplexed on-line instrument starting from DNA templates to their primary sequences has been demonstrated based on multiplexed microfluidics and capillary array electrophoresis. The instrument automatically processes eight templates through reaction, purification, denaturation, preconcentration, injection, separation, and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were utilized to manage flow and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an eight-capillary array in a hot-air thermal cycler. Subsequently, the sequencing ladders are directly loaded into separate size exclusion chromatographic columns operated at approximately 60 degrees C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at approximately 70 degrees C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. The raw data allow base calling up to 460 bp with an accuracy of 98%. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also genetic typing.     

Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis

Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect.     

Use of capillary electrophoresis as a method development tool for classical gel electrophoresis

Capillary electrophoresis (CE) was used to optimize the buffer pH, ionic strength and sulfated cyclodextrin concentrations for enantiomeric separation of piperoxan. These enantioseparation conditions were then applied to a classical gel electrophoresis system. Binding constants of the sulfated beta-cyclodextrin-piperoxan couple were approximated using CE and the effects of organic solvents on the system were also investigated.     

Some properties of a measure of resolution in gel electrophoresis and capillary zone electrophoresis

The most commonly used measure of resolution for chromatographic and electrophoretic separations does not take into account the possibility of there being different amounts of each of the molecular species. A modification of a measure of resolution recently suggested by Aldroubi and Garner (BioTechniques 1992, 13, 620-624) can incorporate this effect explicitly. Their criterion for resolution is based on the time to observe a valley of specified magnitude separating two peaks. We examine how this measure depends on different physically relevant parameters that characterize the system.     

Pluronic copolymer liquid crystals: unique, replaceable media for capillary gel electrophoresis

Liquid crystalline solutions of Pluronic copolymers are versatile alternatives to solutions of entangled, random coil polymers as replaceable media for capillary gel electrophoresis (CGE). Pluronic copolymers are tri-block polymers of poly(ethylene oxide) [(EO)x] and poly(propylene oxide) [(PO)y] with the general formula (EO)x(PO)y(EO)x. Large micelles form in aqueous solutions in which central, hydrophobic cores of (PO)y segments are surrounded by "brushes" of hydrated (EO)x tails. Solutions of Pluronic F127 (BASF Performance Chemicals) in a concentration range of about 18-30% are liquids at refrigerator temperatures (< or = 5 degrees C) and are easily introduced into capillaries. A self-supporting, gel-like liquid crystalline phase is formed as the temperature is raised to > or = 20 degrees C. This liquid crystalline phase consists of spherical micelles with diameters of 17-18 nm which pack with local cubic symmetry. CGE in Pluronic F127 liquid crystals separates species within several chemical classes as varied as nucleoside monophosphates and organic dyes, oligonucleotides of 4-60 nucleotides, DNA fragments of 50-3000 base pairs (bp), and supercoiled plasmid DNAs of 2000-10,000 bp. Mechanisms of molecular sieving in polymer liquid crystals must differ in fundamental ways from separations in random polymer gels because molecules move around uncrosslinked obstacles that are larger than the smallest dimensions of typical analytes. Molecular sieving in Pluronic liquid crystals is envisioned to occur as molecules squeeze between hydrated (EO)x strands of micelle brushes, or through brushtips and interstitial spaces between micelles. Small molecules such as nucleotides appear to separate by a different mechanism involving partitioning between hydrophilic and hydrophobic environments. This process is termed "hydrophobic interaction electrophoresis". The unique structures of Pluronic copolymers and their liquid crystalline phases provide new challenges and opportunities in separations science.     

Micropreparative capillary electrophoresis of DNA by direct transfer onto a membrane

We have developed a new technique for the collection of DNA fragments separated by capillary electrophoresis, by direct transfer from the capillary outlet to a positively charged membrane. Transfer and post-run detection of two different nonradioactively labeled DNA standards, ranging in size from 150 bp to 2 kbp and 120 bp to 23 kbp are presented, and discussed. Capillary electrophoresis with direct blotting presents several advantages over the blotting from gels: the separation is faster and requires less manual steps, the resolution is higher, and each DNA fragment is collected into a very concentrated spot on the membrane due to the small surface of the capillary outlet and to a design of the collection device inducing a refocusing of field lines across the hybridization membrane. Therefore, very small amounts of DNA (in the pg range) can be detected. This fraction collection makes further analysis of the sample possible, e.g. by hybridization, thus suppressing one of the major present limitations of the capillary electrophoresis technique for DNA analysis.     

High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing

Skeletal development of transgenic mice with a type II collagen mutation was analyzed and compared with wild-type littermates. The single base substitution in Col2a1 resulted in a glycine to serine mutation within the helical domain and corresponded to one previously identified in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:4583-4587). Skeletal staining of embryos from 14.5 through 18.5 days of gestation demonstrated a dwarf phenotype in the transgenic embryos, most notably short limb bones and vertebral column that was first detected at 15.5 days post-coitus. In addition to the reduced length, the extent of ossification was less in the transgenic mice. The architecture of the long bone growth plate was abnormal in the transgenic tissue, in particular there was no discernible proliferative zone. There were few stacks of characteristically flattened cells and the overall length of the growth plate in the mutant embryos was reduced. At the ultrastructural level, there were fewer collagen fibrils present in the transgenic mouse cartilage compared to that of wild-type littermates. Ultrastructural localization of collagen types II, IX and XI revealed a similar pattern between the transgenic and wild-type pups, suggesting that the collagen fibrils present in the matrix of littermates with both phenotypes had a similar composition. Skeletal analysis and cartilage histochemistry indicated that effect of the type II collagen mutation was to reduce the density of the collagen fibrils within the cartilage matrix which was associated with delayed bone formation and resulted in a short-limbed phenotype.     

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.     

Base stacking: pH-mediated on-column sample concentration for capillary DNA sequencing

An on-column sample concentration method for capillary-based DNA sequencing was studied. This base-stacking method allows direct injection of unpurified products of dye-primer sequencing reactions onto capillaries without any pretreatment. On-column concentration of DNA fragments is achieved simply by electrokinetic injection of hydroxide ions. A neutralization reaction between these OH- ions and the cationic buffer component Tris+ results in a zone of lower conductivity, within which field focusing occurs. DNA fragments are concentrated at the front of this low-conductivity zone. With sample injection times as long as 360 s at 50 V/cm, resolution could still be restored by the stacking process. Using a 36/47-cm-long uncoated capillary, with poly(dimethylacrylamide) as the separation matrix, and electric field of 160 V/cm, a resolution of at least 0.5 could be generated for fragments up to 650 nucleotides long. Both resolution and signal strength are excellent relative to conventional injection of highly purified samples. No significant degradation of the capillary performance was observed over at least 20 sequencing runs using this new sample injection methods.     

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.