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.     

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.     

Finding a universal low viscosity polymer for DNA separation (II)

When investigating the use of different polymers for capillary electrophoresis we found that poly-N,N-dimethylacrylamide (pDMA) has a very low viscosity compared to other polymers of comparable molecular mass and resolving power. This makes it a potentially useful matrix for DNA separation in multi-capillary electrophoresis, where short cycle times or low pressure for matrix replacement are preferred. We have characterized this matrix by systematic studies on concentration, chain length and field strength dependence. It is shown that pDMA performs well for the separation of oligonucleotides and double-stranded DNA fragments. Together with the application of DNA sequencing, pDMA is a universal polymer for the separation of biological macromolecules.     

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.     

Finding a universal low-viscosity polymer for DNA separation

We have investigated the viscosity of different commercially available polymers in solution and found that dextran has a low viscosity compared to other polymers of comparable molecular weight and resolving power. This makes it a potentially useful matrix for DNA separation in capillary electrophoresis, where either short time or low pressure are preferred for matrix replacement. We showed that dextran performs well for the separation of oligonucleotides and double-stranded DNA fragments. Together with the well-known application for protein separation, this makes dextran a universal polymer for the separation of biological macromolecules.     

Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts

Techniques have been developed for extracting, amplifying and directly sequencing mitochondrial DNA (mtDNA) from human hair shafts. The hair shaft is ground in a glass micro-tissue grinder, and the DNA is extracted with organic solvent and purified by filtration. The filtrate subsequently provides the mtDNA template for the PCR. The two hypervariable segments of the mtDNA control region are amplified in four separate reactions. After a purification step to remove unincorporated PCR primers, amplified products are quantitated by capillary electrophoresis and subjected to cycle sequencing. The products are separated and analyzed on an automated DNA sequencer. The mtDNA sequences from the hair shaft match the mtDNA sequences from blood samples taken from the same donor.     

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.     

Characterization of dye-induced mobility shifts affecting DNA sequencing in poly(ethylene oxide) sieving matrix

The influence of three classes of fluorescence labels including dipyrrometheneboron difluoride (BODIPY), energy transfer (ET) and conventional fluorescein and rhodamine (ABI), on DNA sequencing has been examined with laser-induced fluorescence detection and poly(ethylene oxide)-filled capillary electrophoresis. DNA sequencing fragments were generated by dye-labeled primer cycle-sequencing reactions in a hot-air thermal cycler. A parameter, relative-induced shift, was introduced to quantify the uniformity of electrophoretic mobilities of these fragments. BODIPY was found to have the smallest, but nonzero, effect for dye-induced nonuniformity. Although ET dyes provided the highest sensitivity due to their unique spectroscopic properties, they were found to lack photostability compared to BODIPY and ABI dyes. Characterization also brings out some important tips for selecting the suitable dye set for the two-channel ratio-based DNA base-calling method.     

The free solution mobility of DNA

The free solution mobility of DNA has been measured by capillary electrophoresis in the two buffers most commonly used for DNA gel electrophoresis, Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE). The capillaries were coated with polymers of either of two novel acrylamide monomers, N-acryloylaminoethoxyethanol or N-acryloylaminopropanol, both of which are stable at basic pH and effectively eliminate the electroendosmotic mobility due to the capillary walls. The free solution mobility of DNA in TAE buffer was found to be (3.75 +/- 0.04) x 10(-4) cm2 V-1 s-1 at 25 degrees C, independent of DNA concentration, sample size, electric field strength, and capillary coating, and in good agreement with other values in the literature. The free solution mobility was independent of DNA molecular weight from approximately 400 base pairs to 48.5 kilobase pairs, but decreased monotonically with decreasing molecular weight for smaller fragments. Surprisingly, the free solution mobility of DNA in TBE buffer was found to be (4.5 +/- 0.1) x 10(-4) cm2 V-1 s-1, about 20% larger than observed in TAE buffer, presumably because of the formation of nonspecific borate-deoxyribose complexes.     

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.     

A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocol

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.     

A discontinuous buffer system increasing resolution and reproducibility in DNA sequencing on high voltage horizontal ultrathin-layer electrophoresis

A novel discontinuous buffer system for DNA sequencing based on horizontal ultrathin-layer gel electrophoresis is described. The optimized system, named unbuffered stacking gel/discontinuous borate EDTA-buffer system, is composed of a 0.5 mm thick stacking gel, where standard sequencing reactions (1 microL volume) are easily loaded, and a 50 microns ultrathin running gel, where DNA fragments are separated. The novel discontinuous buffer system allows for sample concentration and efficient injection from the stacking gel into the capillary slab gel. Increased resolution, assessed by autoradiography, can be achieved within 25 min running time already over a 10.1 cm distance from the gel slot compared to the conventional gel system. An advantage of the new system is the capacity to resolve compressions in GC-rich regions, usually causing migrating artifacts in standard gels. The described system affords a major improvement in speed, resolution and reproducibility in DNA sequencing.     

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.     

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.     

Analysis of disease-causing genes and DNA-based drugs by capillary electrophoresis. Towards DNA diagnosis and gene therapy for human diseases

Rapid progress in the Human Genome Project has stimulated investigations for gene therapy and DNA diagnosis of human diseases through mutation or polymorphism analysis of disease-causing genes and has resulted in a new class of drugs, i.e., DNA-based drugs, including human gene, disease-causing gene, antisense DNA, DNA vaccine, triplex-forming oligonucleotide, protein-binding oligonucleotides, and ribozyme. The recent development of capillary electrophoresis technologies has facilitated the application of capillary electrophoresis to the analysis of DNA-based drugs and the detection of mutations and polymorphism on human genes towards DNA diagnosis and gene therapy for human diseases. In this article the present state of studies on the analysis of DNA-based drugs and disease-causing genes by capillary electrophoresis is reviewed. The paper gives an overview of recent progress in the Human Genome Project and the fundamental aspects of polymerase chain reaction-based technologies for the detection of mutations and polymorphism on human genes and capillary electrophoresis techniques. Attention is mainly pad to the application of capillary electrophoresis to polymerase chain reaction analysis, restriction fragment length polymorphism, single strand conformational polymorphism, variable number of tandem repeat, microsatellite analysis, hybridization technique, and monitoring of DNA-based drugs. Possible future trends are also discussed.     

Measurement of whole blood phagocyte chemiluminescence in the Wistar rat

The utility of capillary electrophoresis in the study of DNA-protein binding is demonstrated, using the minimal DNA binding domain of the onco-protein c-Myb (R2R3) and a specific target DNA sequence as a model system. The capillary electrophoresis method is based on simple UV detection at 260 nm with a linear polymer buffer and a coated capillary, and requires no labeling or derivatization of the DNA. A specific protein-DNA complex is observable as a retarded peak, which increases with increasing protein concentration with a corresponding reduction in the free DNA peak. With DNA and protein preparations of known concentrations, a test for sequence-specific binding can be completed within 10 min.     

Sanger DNA-sequencing reactions performed in a solid-phase nanoreactor directly coupled to capillary gel electrophoresis

A miniaturized, solid-phase nanoreactor was developed to prepare Sanger DNA-sequencing ladders which was directly interfaced to a capillary gel electrophoresis system. A biotinylated fragment of the rat brain actin gene (1 kbp) was amplified by PCR and attached to the interior wall of an (aminoalkyl)silane-derivatized fused-silica capillary tube via a biotin/streptavidin/biotin linkage. Coverage of the capillary wall with the biotinylated DNA averaged 77 +/- 10%. Stability of the anchored template under pressure (33 nL/s) and electroosmotic flows (11.3 nL/s) were favorable, requiring rinsing for > 150 h to reduce the surface coverage by only 50%. In addition, the immobilized template was stable toward temperatures required for preparing sequencing ladders, even under cycling conditions. Standard Sanger dideoxynucleotide termination performed in a large-volume (approximately 8 microL) solid-phase reactor using the thermally stable polymerase enzymes Taq and Vent and the polymerases T7 and Bst with off-line slab gel electrophoresis and autoradiographic detection indicated that acceptable fragment generation was achieved only in the case of the thermally stable polymerases. Banding was not apparent for T7 and Bst since all reagents were inserted into the column in a single plug at the beginning of the reaction. A small volume reactor (volume approximately 62 nL) was then used to perform DNA polymerase reactions and was coupled directly to a capillary gel column for separation. The capillary reactor was placed inside a thermocycler to control the temperature during chain extension and was directly connected to the gel column via zero dead volume fused-silica connectors. The complementary DNA fragments generated (C-track only) in the reactor were denatured using heat and directly injected onto the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. Extension and single-base separation resolution of the C-track, which was directly injected onto the gel column, was estimated to be > 450 bases from the primer annealing site with plate numbers ranging from 1 x 10(6) to 2 x 10(6)/m.     

Integrated chip-based capillary electrophoresis

Integrated capillary electrophoresis (ICE) is emerging as a new analytical tool allowing fast, automated, miniaturized and multiplexed assays, thus meeting the needs of the pharmaceutical industry in drug development. The current state-of-the-art of ICE is described with an emphasis on the choice of the support material (glass or polymeric materials), electrokinetic fluid handling, and injection and detection issues. Strategies and chip designs for pre- or post-column derivatization, DNA sequencing, on-line PCR analysis, on-chip enzymatic sample digestion, fraction isolation, and immunoassays are presented. The review concludes with a brief outlook.     

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.