Capillary zone electrophoresis with on-line blotting for separation and detection of 32P-postlabelled DNA adducts

A new technique for the detection of ³²P-postlabelled DNA adducts separated by capillary electrophoresis was developed. By direct transfer from the capillary outlet to a positively charged moving filter paper, eluted radioactive peaks can be quantified using a phosphor imaging detector. With this method it is possible to separate DNA adducts from different carcinogens after ³²P-postlabelling of the modified and unmodified nucleotides with high sensitivity approaching 1 adduct per 10⁹ nucleotides.     

Detection of mutations in exon 8 of TP53 by temperature gradient 96-capillary array electrophoresis

Various capillary electrophoresis applications have increasingly been utilized in mutation detection. Separation of two species is either based on secondary structure or differences in melting of DNA due to the mutation. Detection of the mutant is based on its mobility difference in the sieving matrix. We have adapted a regular 96-capillary sequencing instrument, the MegaBACE 1000, for mutation detection based on thermodynamic stability and mobility shift during electrophoresis. Denaturation of the lower melting domain of the DNA was achieved with a gradually decreasing temperature gradient in combination with a chemical denaturant. Samples were analyzed for mutants in exon 8 of the TP53 genefrom tumor samples and controls. Genomic DNA was PCR-amplified with one fluorescein labeled primer and one GC-clamped primer, diluted in water, and analyzed by temperature gradient 96-capillary array electrophoresis. Tumor samples and PCR reconstruction experiment samples were resolved by capillary gel electrophoresis under appropriate temperature gradient denaturing conditions. Ninety-six samples were analyzed in one run, with an analysis time of 30 min and a sensitivity to detect mutated alleles in wild-type background down to 0.4%. The technique proved to be robust, in that the gradient compensatesfor temperature differences within the capillary chamber; thus, each capillary will pass through the optimal separating conditions around the theoretical melting temperature for TP53 exon 8, separating homoduplexes and heteroduplexes. This technique is applicable to any sequence previously analyzed by DNA melting gel techniques or sequences harboring iso-melting domains of 100-120 bp.     

High-efficiency DNA ligation for clamp attachment without polymerase chain reaction

We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA. Our approach can be added to a constant denaturant capillary electrophoresis (CDCE)-based method of analyzing rare point mutants at fractions as low as 10(-6); such mutants appear as small copy numbers in the initial samples. This CDCE-based method alone is applicable to only those DNA sequences juxtaposed with an internally occurring clamp of a higher melting temperature in genomic DNA. Since such sequences represent 9% of the human genome, the addition of clamp ligation significantly increases the scanning range for the human genome without reducing the initial mutant copy numbers. Furthermore, clamp ligation/attachment without PCR prevents PCR-created mutants from interfering with rare mutational analysis. In addition to those applications seeking high-efficiency DNA ligation, our approach can be generally applied to ligation of restriction ends.     

Identification of in vivo mutations in exon 5 of the human HPRT gene in a set of pooled T-cell mutants by constant denaturant capillary electrophoresis (CDCE)

Constant denaturant capillary electrophoresis (CDCE), based on co-operative DNA melting equilibria, has the resolving power to separate single nucleotide mutants from wild type sequences. We used this technique to study mutations in a 70-bp isomelting domain of the human HPRT gene, which included the entire exon 5 and its flanking splice donor and acceptor sites. Pooled samples of 6-thioguanine selected T-cell clones from 51 healthy donors representing a total of approximately 1000 individual HPRT mutants were analysed. Slow moving peaks from the heteroduplex part of the CDCE electropherograph were collected and subjected to a second round of PCR and CDCE analysis, followed by DNA sequencing. Five independent mutations were detected. Four were splicing errors; one insertion of CC and two G-->A transitions in the splice donor site of intron 5, and one G-->C transversion in the splice acceptor site of intron 4. The fifth mutation was a missense transversion, T389>G. A reconstruction experiment, in which DNA with known mutation was mixed with wild type DNA, showed the sensitivity of mutation detection to be better than 1:100 under the conditions used in this study. These results demonstrate the high sensitivity of the CDCE-method for mutation screening.     

Identification of point mutations in mixtures by capillary electrophoresis hybridization.

We have developed a rapid method for unambiguous identification and mutant fraction determination of individual mutants in mixtures of DNA sequence variants each differing by one or a few nucleotides. This method has applications to such diverse areas as interpretation of mutational spectra, screening of populations for polymorphisms and identification of species in environmental mixtures. In our approach, a mixture of unknown sequences labeled with a fluorescent dye is combined with a set of predetermined sequences (standards) representing the variants to be assayed. Labeling the standards with another dye allows the two sets of variants to be measured independently. Using constant denaturing capillary electrophoresis, the sequence variants are separated as individual peaks on the basis of differential melting equilibria. The unknown sequence variants are initially identified based on co-migration with particular standards. This preliminary identification is verified by hybridization of the unknown variants with the co-migrating standards within the capillary. We demonstrate the use of capillary electrophoresis hybridization to dissect complex mutational spectra of human cells in culture.     

A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA

Knowledge of the kinds and numbers of nuclear point mutations in human tissues is essential to the understanding of the mutation mechanisms underlying genetic diseases. However, nuclear point mutant fractions in normal humans are so low that few methods exist to measure them. We have now developed a means to scan for point mutations in 100 bp nuclear single copy sequences at mutant fractions as low as 10^–6. Beginning with about 10⁸ human cells we first enrich for the desired nuclear sequence 10 000-fold from the genomic DNA by sequence-specific hybridization coupled with a biotin–streptavidin capture system. We next enrich for rare mutant sequences 100-fold against the wild-type sequence by wide bore constant denaturant capillary electrophoresis (CDCE). The mutant-enriched sample is subsequently amplified by high fidelity PCR using fluorescein-labeled primers. Amplified mutant sequences are further enriched via two rounds of CDCE coupled with high fidelity PCR. Individual mutants, seen as distinct peaks on CDCE, are then isolated and sequenced. We have tested this approach by measuring N-methyl-N ′-nitro-N-nitrosoguanidine (MNNG)-induced point mutations in a 121 bp sequence of the adenomatous polyposis coli gene (APC) in human lymphoblastoid MT1 cells. Twelve different MNNG-induced GC→AT transitions were reproducibly observed in MNNG-treated cells at mutant fractions between 2 × 10^–6 and 9 × 10^–6. The sensitivity of this approach was limited by the fidelity of Pfu DNA polymerase, which created 14 different GC→TA transversions at a mutant fraction equivalent to ~10^–6 in the original samples. The approach described herein should be general for all DNA sequences suitable for CDCE analysis. Its sensitivity and capacity would permit detection of stem cell mutations in tissue sectors consisting of ~10⁸ cells.     

Ligation of high-melting-temperature ‘clamp’ sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10 000 and 5/100 000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 × 10^–4. Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10^–6 using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (~100 bp) juxtaposed with a ‘clamp’, a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent ~9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 × 10^–5 in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 × 10⁴ turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10⁵ persons.     

High resolution melting analysis for gene scanning

High resolution melting is a new method of genotyping and variant scanning that can be seamlessly appended to PCR amplification. Limitations of genotyping by amplicon melting can be addressed by unlabeled probe or snapback primer analysis, all performed without labeled probes. High resolution melting can also be used to scan for rare sequence variants in large genes with multiple exons and is the focus of this article. With the simple addition of a heteroduplex-detecting dye before PCR, high resolution melting is performed without any additions, processing or separation steps. Heterozygous variants are identified by atypical melting curves of a different shape compared to wild-type homozygotes. Homozygous or hemizygous variants are detected by prior mixing with wild-type DNA. Design, optimization, and performance considerations for high resolution scanning assays are presented for rapid turnaround of gene scanning. Design concerns include primer selection and predicting melting profiles in silico. Optimization includes temperature gradient selection of the annealing temperature, random population screening for common variants, and batch preparation of primer plates with robotically deposited and dried primer pairs. Performance includes rapid DNA preparation, PCR, and scanning by high resolution melting that require, in total, only 3 h when no variants are present. When variants are detected, they can be identified in an additional 3 h by rapid cycle sequencing and capillary electrophoresis. For each step in the protocol, a general overview of principles is provided, followed by an in depth analysis of one example, scanning of CYBB, the gene that is mutated in X-linked chronic granulomatous disease.     

Analysis of mutational spectra by denaturing capillary electrophoresis

The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene-disease associations and microbial populations. Detection limits are about 5 x 10(-3) (mutants copies/total copies) but can be as low as 10(-6) (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.     

Determination of genomic 5-hydroxymethyl-2’-deoxycytidine in human DNA by capillary electrophoresis with laser induced fluorescence

Recent studies reported the presence of 5-hydroxymethylcytosine (5 hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine.

Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5 hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A double stranded DNA fragment containing a defined number of 5 hmdC residues was used for peak assignment, to establish separation conditions and to determine the limit of detection (LOD). The method yielded a LOD for 5 hmdC of 0.45 amol, which is equivalent to approximately to one 5 hmdC per 4,000 normal nucleotides (0.025%) using 1 μg of DNA as the matrix.

By applying the calibrated assay to the analysis of various DNAs we show that 5 hmdC is present in human tissue and human cancer cell lines. We demonstrate that by using CE-LIF DNA can be analyzed in one run for both methylation and hydroxymethylation of cytosine with high sensitivity and accuracy.     

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) × 10⁻⁴ cm² V⁻¹ s⁻¹ at 25°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 ∼ 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) × 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. © 1997 John Wiley & Sons, Inc. Biopoly 42: 687–703, 1997     

Mutation detection using Surveyor nuclease

We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.     

Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis

The ability to detect DNA sequence heterogeneity quickly and reliably is becoming increasingly important as more genes involved in disease processes are discovered. We have assessed the ability of a high pressure liquid chromatography technique (HPLC) termed temperature-modulated heteroduplex analysis (TMHA) to detect a collection of 20 point mutations distributed throughout a 279 base pair fragment spanning the exon 8 region of the human HPRT gene. All mutant/wild type heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved from their parental homoduplexes. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA and DGGE are measuring similar melting characteristics in heteroduplex molecules. TMHA appears to be a robust approach for detecting and/or purifying a wide variety of mutations in a defined region of DNA, provided that the melting characteristics of the fragment under study are carefully considered.     

Clustering of mutant mitochondrial DNA copies suggests stem cells are common in human bronchial epithelium

Tissue maintenance stem cells, as opposed to transition and/or terminal cells in the epithelium, are possible progenitor cells for human tumors, but little is known about their frequency in human tissues. It occurred to us that the colonies of mutants that should be created when a stem cell mutates and transmits the rare mutation to its descendent transition and terminal cells should, given a quantitative mutation assay, define the average number of cells in a maintenance turnover unit and permit calculation of stem cell number. To test this concept we used a combination of high fidelity PCR and constant denaturant capillary electrophoresis to enumerate mitochondrial point mutations and define their number and distribution among multiple small samples of approximately one million cells containing about 400 million copies of mitochondrial DNA. The bulk of the data were best explained by a model in which most stem cells, defined here as long-lived cells, give rise to colonies of approximately 8-128 cells. In addition, we found that about 1.5% of colonies contained hundreds or even thousands of homoplasmic mutant cells. These expanded turnover units suggest the bronchial epithelium may contain large clusters of cells with mutations, and possibly phenotypic alterations as well.     

Interaction of 2‐Chloro‐N10‐Substituted Phenoxazine with DNA and Effect on DNA Melting

Five N¹⁰‐substituted phenoxazines having different R groups and –Cl substitution at C‐2 were found to bind to calf –thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA‐ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid _PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.     

Translation of mitochondrial RNA from yeast cytoplasmic petite mutants in anE. coli cell-free system

Mitochondrial RNA from two cytoplasmic ?^? mutants ofS. cerevisiae, which have kept the mitochondrial DNA segment including the ATPase-oligomycin resistance-conferring gene, stimulates protein synthesis in anE. coli cell-free system. SDS-acrylamide gel electrophoresis of the protein product revealed one major peak and two minor ones with apparent molecular weights of around 11,000, 13,500 and 17,000 respectively. The effect is specific since no stimulation is observed with RNA from a ?^? mutant devoid of detectable mitochondrial DNA. These results are interpreted to mean that the mitochondrial DNA of these mutants codes for anin vitro translatable mRNA.     

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, antisene 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 paid 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.     

Unexpected behavior of H2Kb mutant DNAs in denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is based upon the different melting behaviors of DNA molecules in a chemical denaturant gradient according to their sequences. This technique has recently become a widespread tool to detect mutations. The introduction of a GC-clamp enables the detection of most single base differences between two DNA molecules. As a test system we have applied the polymerase chain reaction (PCR) in combination with DGGE to detect a number of mutations in the mouse H2Kb DNA sequence. A wide variety of spontaneous in vivo mutations of this haplotype have been reported in the C57BL/6J mouse strain and are clustered in the alpha 1 and alpha 2 domains. The combination of PCR and DGGE revealed almost all base changes present in the H2Kb mutants used. However, most of the PCR products of these mutants showed melting behavior which is not easily predicted. We suggest that in addition to current simple theory, which considers that the migration of a DNA molecule in a denaturing gradient depends primarily on its initial melting behavior, additional factors such as secondary structure in partially melted molecules may play a role and can be used to detect mutations.     

Direct measurement of mutational spectra in humans

By combining high fidelity in vitro DNA amplification and mutant DNA sequence separation by denaturing gradient gel electrophoresis, we are able to directly observe mutational hotspots in human genomic DNA. Our technological development has progressed through the stage of identifying mutant sequences in independently derived, 6-thioguanine-resistant human B cells. We are now analyzing uncloned, complex populations derived from several thousand 6-thioguanine-resistant cells and report preliminary data concerning the mutational spectra of benzo[a]pyrene diol epoxide and ultraviolet light in exon 3 of the hypoxanthine-guanine phosphoribosyltransferase gene. In addition, the approach appears to be general for any gene sequence for which a means to select mutants exists. The more global need to eliminate phenotypic selection is, however, our primary impetus. Our analysis leads us to conclude that no known in vitro DNA polymerase has sufficient fidelity to permit direct observation of unselected mutants. Therefore, an additional change in technology will be necessary to observe nonselected mutant DNA sequences at the low frequencies found in human tissues.