DNA Capillary Electrophoresis Research Articles

Parallel molecular genetic analysis

We describe recent progress in parallel molecular genetic analyses using DNA microarrays, gel-based systems, and capillary electrophoresis and utilization of these approaches in a variety of molecular biology assays. These applications include use of polymorphic markers for mapping of genes and disease-associated loci and carrier detection for genetic diseases. Application of these technologies in molecular diagnostics as well as fluorescent technologies in DNA analysis using immobilized oligonucleotide arrays on silicon or glass microchips are discussed. The array-based assays include sequencing by hybridization, cDNA expression profiling, comparative genome hybridization and genetic linkage analysis. Developments in non microarray-based, parallel analyses of mutations and gene expression profiles are reviewed. The promise of and recent progress in capillary array electrophoresis for parallel DNA sequence analysis and genotyping is summarized. Finally, a framework for decision making in selecting available technology options for specific molecular genetic analyses is presented.

A new separation medium for DNA capillary electrophoresis: self-assembly behavior of Pluronic polyol E 99P 69E 99 in 1X TBE buffer

The unique sol–gel transition property of E 99P 69E 99 (with E and P being poly(ethylene oxide) and poly(propylene oxide), respectively), a Pluronic polyol F127, could become a useful separation medium in the miniaturization and automation of capillary electrophoresis (CE). Static light scattering (SLS), dynamic light scattering (DLS), and small-angle X-ray scattering were used to study the solution property and the gel-like structures of F127 in a CE buffer (1X TBE) for DNA separation. F127 in the CE forms core-shell micelles with a closed-association process. The critical micelle concentration (cmc) decreases and the micellar association number increases with increasing temperature. At larger solution concentrations, small-angle X-ray scattering results showed that the gel-like structure formed by close packing of the micelles had a face centered cubic (fcc) lattice. In the gel-like region the association number and also the micellar size were not affected by concentration changes. With F127 solution as a separation medium, CE could be performed successfully with a detection length of only 4 cm for separation of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) fragments.

A three-wavelength labeling approach for DNA sequencing using energy transfer primers and capillary electrophoresis.

Capillary electrophoresis DNA sequencing has been accomplished by using four different energy transfer primers and three fluorescence detection channels. Methods have also been developed to deconvolve the three-color data into the four base concentrations. The nonnegative least squares and model selection method resulted in the best accuracy. The three-color data were compared to sequencing data obtained using four detection channels and four energy transfer primers. The average accuracy rates obtained over three 500 base M13mp18 runs using three-color coding were 96% including 18 uncallable compressions and 99.6% if these compressions are excluded. The average accuracy rate obtained using four-color coding was 96.3% including 18 uncallable compressions and 99.9% if these compressions are excluded. Although it is unlikely that three-color schemes will replace four-color sequencing, these methods have exposed basic concepts that will be useful in the development of higher-order multiplex coding methods for DNA analysis.

Maternal Identification from Skeletal Remains of an Infant Kept by the Alleged Mother for 16 Years with DNA Typing

This is a case study concerning maternal identification by DNA typing at various loci. An infant skeleton was found in the alleged mother's apartment after it was kept for 16 years. We obtained the skeletal remains as well as saliva stains from the alleged mother. DNA typing was conducted for three loci in the HLA class II region (HLA-DQA1, -DPB1, and DRB1), five loci with the AmpliType PM kit (LDLR, GYPA, HBGG, D7S8, and GC), five STR loci (LPL, vWA, F13B, TH01, and TPOX) and D-loop region in mtDNA for maternal identification. Sex determination was accomplished using fluorescent DNA capillary electrophoresis typing. Approximately 5 ng of human DNA was recovered from 1 g of femur bone retrieved from the infant skeletal remains. The probability of two unrelated Japanese sharing the same genotypes was estimated as 7.2 x 10(-11). The combined probability of exclusion that an individual is not the mother was also calculated at 0.998. We therefore conclude that the skeleton is from a female infant, and that there is no inconsistency in the claim that the infant was a daughter of the alleged mother.

Band broadening during dc and field inversion capillary electrophoresis of lambda DNA in dilute hydroxyethylcellulose solutions.

Capillary electrophoresis of lambda double-stranded (ds) DNA (48.5 kbp) in dilute hydroxyethylcellulose (HEC) solutions shows band spreading that cannot be explained by diffusion alone. Dispersion and asymmetry factors of lambda ds-DNA bands were measured as functions of capillary length, HEC concentration and field strength. Band spreading and asymmetry can be explained by a recently developed model in which the dominant contribution is assumed to be dispersion in DNA-HEC disentanglement times. Bandwidth reduction using square-wave field inversion was also investigated. It is proposed that correlation of DNA motion is the source of band narrowing during field inversion.

Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis

Capillary electrophoresis has become a powerful analytical tool for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. The use of replaceable polymer solutions increases the lifetime of the capillary, improves the repeatability of the migration times and enables pressure injection. When pressure injection is used, rather than electrokinetic injection, it is assured that a representative part of the sample is introduced into the capillary. The possible lower resolution, which is a side effect of the pressure injection, can be made up for by an increase of selectivity. Using fluorescent labels, it is possible to detect DNA with a fluorescence detector under both native conditions in double-stranded (ds) form, and under denaturing conditions in single-stranded (ss) form. When DNA is separated in its ss form, as is necessary for DNA sequencing, we observe an increased selectivity compared to separation of that sample in its ds form. In this work, we exploited this increased selectivity for the analysis of denatured PCR products. It was found that DNA separated in the ss form yields superior separation; that is, a given analysis can be achieved with the same resolution in a shorter separation time compared to dsDNA. Therefore, it is advisable to separate DNA in the ss form if high resolution, size dependent separation is required. The enhanced resolution achieved with DNA migrating in the ss form enabled the separation of allelic ladders of short tandem repeats with a difference of 4 base pairs in the 200 base pair range, with separation times no longer than 6 min.

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

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

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

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

Pseudo-coulmetric loading in capillary electrophoresis DNA sequencing

While injection volumes in capillary electrophoresis are typically in the nanoliter range, it is difficult to physically prepare and manipulate samples much smaller than a microliter. As a result, only a small fraction of the analyte contained with the sample volume is transferred to the capillary. This problem is particularly acute in DNA sequencing applications, where on-column stacking is difficult and where the sequencing sample is relatively expensive to prepare. We report a method that transfers 75% of the DNA contained within a 3 μl sample onto a capillary for DNA sequencing. This method relies on the use of very low ionic strength formamide to resuspend the DNA after an ethanol precipitation. The use of low ionic strength formamide achieves two tasks. First, it produces a very high resistance sample, which increases the voltage drop across the sample and decreases the field across the capillary. This electric field manipulation ensures that DNA fragments do not migrate down the capillary during the loading process, allowing long injection periods without excessive band-broadening. Second, the low ionic strength of the formamide increases the transference number of the DNA; more of the current passing through the injection tip of the capillary is carried by DNA fragments. In the limit of complete elimination of impurity ions from the loading solvent, current passing through the sample is carried only by DNA fragments and loading becomes a coulometric process.

Automated polymerase chain reaction product sample preparation for capillary electrophoresis analysis.

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.

Electrophoresis of DNA in novel thermoreversible matrices.

We demonstrate the feasibility of temperature-sensitive polymers as novel matrices for capillary and slab electrophoresis of DNA. These matrices combine the case-of-use of agarose with resolution properties of polyacrylamide. Two classes of matrices are used: (i) aqueous suspensions of gel microspheres and (ii) solutions containing uncross-linked temperature-sensitive polymers. When heated, the viscosity of these solutions drops dramatically because of the phase transition behavior of these polymers, and therefore these formulations are easy to pour or load. Results are presented for separation of double-stranded DNA fragments (< 2000 bp) in capillary, tube, and slab electrophoresis. Preliminary results are also presented for separation of single-stranded sequencing fragments by capillary electrophoresis. In the tube format, good resolution was obtained for phi X174/HaeIII fragments. In the slab format, excellent resolution of double-stranded DNA was obtained, including simultaneous separation of a 10 bp ladder up to 150 base pairs. In the capillary format, pBR322/MspI fragments were completely resolved, and single-base resolution of sequencing fragments was obtained up to 150 bases. We believe that temperature-sensitive polymers represent a new and promising class of electrophoretic media.

Design and Synthesis of Fluorescence Energy Transfer Dye-Labeled Primers and Their Application for DNA Sequencing and Analysis

We have designed and synthesized fluorescent oligonucleotide primers having improved fluorescence and electrophoretic properties by exploiting the concept of resonance fluorescence energy transfer (ET). These primers carry a fluorescein derivative at the 5′ end as a common fluorescence donor and other fluorescein and rhodamine derivatives attached to a modified thymidine within the primer sequence as acceptors. These primers all have strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The fluorescence emission intensity of the ET primers increases as the spacing between the donor and accepters is increased, and of the spacings studied the strongest fluorescence was observed when the number of nucleotides between the donor and acceptors is 10. The electrophoretic mobilities of the primers were also found to be a function of the spacing between the donor and the acceptors, and the mobilities of the single base extension DNA fragments generated with primers F10F, F10J, F10T, and F10R were closely matched. With excitation at 488 nm, the fluorescence of the four optimized ET primers (F10F, F10J, F10T, and F10R) is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragments generated with the four ET primers allow four-color DNA sequencing on a capillary electrophoresis DNA sequencer using a single laser line at 488 nm for excitation and without applying mobility shift adjustments. With single-stranded M13mp18 DNA as the template, a typical run with the ET primers on a commercial sequencer provided DNA sequences with 99-100% accuracy in the first 500 bases using 8-fold less DNA template than that typically required using T7 DNA polymerase.

Systematic study of field and concentration effects in capillary electrophoresis of DNA in polymer solutions

A systematic study of the separation of double-stranded DNA in hydroxypropylcellulose (HPC) with a molecular mass of 10 6 was undertaken, using a variety of concentrations (from 0.1 to 1%) and different electric fields (from 6 to 540 V/cm). The data show that a high polymer concentrations (⩾0.4%) and low fields, the separation mechanism is similar to that occurring in gels. The results are in good agreement with theoretical models, and in particular with a recently proposed theory for gels with a pore size smaller than the persistence length of DNA. For more dilute solutions and high fields, however, the separation pattern cannot be explained by existing theories. The existence of an original mechanism was confirmed by the direct observation of the conformation of double-stranded DNA molecules in the polymer solution by fluorescence videomicroscopy. Practical conclusions for the capillary electrophoretic separation of duplex DNA are drawn.

Sequence dependent migration behavior of double-stranded DNA in capillary electrophoresis.

Capillary electrophoresis (CE) with a replaceable linear polyacrylamide (LPA) sieving matrix was used to examine sequence-dependent migration of double-stranded DNA fragments. It has been found that DNA conformational effects were significant under high electric field separations, especially using high resolution matrices. Compared to linear DNA-ladder standards, both anomalously slow and rapid DNA fragments were observed, with the degree of anomalous migration depending on the electric field strength, polymer concentration, column temperature, and background electrolyte (denaturants, sodium and magnesium ions, DNA-intercalating dyes). By selecting a combination of electrophoretic conditions (e.g. 3% T LPA, elevated capillary temperature, lower electric field strength and addition of DNA intercalating dyes), molecular weight dependent separations were closely restored.

Hybridoma cell cultures continuously undergo apoptosis and reveal a novel 100 bp DNA fragment

This report represents an investigation into the nature of apoptosis in hybridoma cultures and its significance to their utilization in biotechnology. To this end DNA fragmentation and capillary electrophoresis of genomic DNA was studied during the culture of two hybridoma cell lines. This indicated that the phenomenon of apoptosis was always present even under normal culture conditions. Two DNA fragments not associated with the typical DNA fragmentation ladder were identified in the two hybridoma cultures: a previously unreported DNA fragment of about 100 bp and a large fragment which may correspond to one reported in the literature (Walker et al., 1993). The small fragment was identified as soon as the early exponential growth phase of culture, while the large fragment appeared only in the latter part of the growth curve when there was marked DNA fragmentation. In addition we present evidence that aurintricarboxylic acid, which inhibits apoptosis in neural cells, permits this process in hybridoma cells at levels below 100 μM. This unusual predisposition of hybridoma cultures to undergo apoptosis and their response to inhibitor of apoptosis may have important implications for approaches to the culture of hybridomas and their utilization for monoclonal production.

Biotechnology core facilities: trends and update

A survey of 128 biotechnology core facilities has provided data on the finances, services, space requirements, and personnel. An average facility had four full-time personnel and 7.5 major instrument systems, and occupied 969 sq. ft. Average total income was $244,000/year, but annual user fee income was only $125,000. Typically, facilities required substantial institutional support or grants. Cost recovery (user fee income divided by total income) averaged 49%. During the last 5 years user fee income, total income, and cost recovery have increased. In-house charges for protein sequencing and peptide synthesis increased approximately 30%, while oligonucleotide synthesis charges decreased by 74%. The costs (charges corrected for subsidy from non-user fee income) for most services did not significantly change, except that oligonucleotide synthesis costs decreased by 25% in 1992. DNA synthesis had the highest throughout per month (116 samples), followed by amino acid analysis (86 samples) and DNA sequencing (67 samples). Other services averaged from 5 to 60 samples. DNA synthesis and purification were the services used by the greatest number of principal investigators. A number of services including DNA sequencing, mass spectrometry, capillary electrophoresis, RNA synthesis, electroblotting, and carbohydrate analysis have been introduced in the last 3 years. Although these services are characterized by high levels of methods development and non-user runs, they are offered by twice the percentage of facilities as in 1989, and are increasingly contributing to facility income.

Capillary electrophoresis of DNA in entangled polymer solutions

Electrophoretic separations of DNA restriction fragments were performed in solutions of hydroxyethylcellulose (HEC) using capillary electrophoresis. Rheological studies confirmed that the entanglement threshold (Φ*) for the solution is ca. 0.003 g/ml, in good agreement with theoretical predictions. A mesh size an order of magnitude smaller than that found in agarose gels 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.

Splendid isolation systems

A benchtop ultracentrifuge, spin columns for the purification of nucleic acids, chromosomal-grade agarose for megabase DNA separations and capillary electrophoresis columns with novel phases – new product ideas in the separation sciences.

Capillary electrophoresis of DNA in agarose solutions at 40°C

DNA fragments ranging from 72 to 1353 bp in length (phi X174 RF DNA/HaeIII) were separated by capillary electrophoresis in 0.3-2.0% solutions of agarose (Sea-Plaque GTG) at 40 degrees C. Liquified agarose above its gelling temperature is easily filled and refilled into capillaries. Its background absorbance at 260 nm was sufficiently low to allow for DNA detection at an estimated DNA load of 13 ng/10 components. Sample injection proceeded at 1 kV for 16 s. The internal capillary diameter was 150 mu, the migration path 27 cm. Migration times varied from 5 to 14 min at 185 V/cm. Potentially, the applicability of capillary electrophoresis in agarose solutions can be expected to extend to the entire size range of DNA, in view of the recent demonstration of kb-sized circular DNA separations in agarose solutions, and those of Mb-sized DNA-agarose complexes in linear polyacrylamide solutions.