Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis

Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild-type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method.     

Review of denaturant capillary electrophoresis in DNA variation analysis

Analyses of germline and somatic single-nucleotide DNA variations are important in both population genetics research and clinical practice. Reliable and inexpensive methods that are flexible and designed for automation are required for these analyses. Present day DNA sequencing technology is too expensive for testing all 22-25 000 human genes in populations genetics studies or in scanning large numbers of tumors for novel mutations. Denaturant capillary electrophoresis (DCE) has the potential to meet the need for large-scale analysis of DNA variants. Several different analyses can be performed by DCE, including mutation analysis, single-nucleotide polymorphism (SNP) discovery in individual and pooled samples, detection of allelic imbalance, and determination of microhaplotypes. Here we review the theoretical background of the method, its sensitivity, specificity, detection limit, throughput, and repeatability in the light of current literature in the field.     

High throughput mutation screening by automated capillary electrophoresis

Molecular diagnosis of complex inherited disorders, population screening of genetic diseases, studies of the genetic basis of variable drug response (pharmacogenetics) as well as discovery and investigation of new drug targets (pharmacogenomics) involve screening for mutations in multiple DNA samples. Furthermore, the development of a third generation of the human genome map, based on single nucleotide polymorphisms (SNPs), requires screening for allelic variants through all of the three billion basepairs in the human genome. Thus, the need for high throughput mutation screening methods is great and is rapidly increasing. Traditional methods for mutation screening often involve slab-gel electrophoresis analyses which are laborious and difficult to automate. However, recent developments in capillary electrophoresis systems for DNA fragment analysis have made fully automated mutation screening possible and have dramatically increased the possible sample throughput. This review describes the recent advances in capillary electrophoresis of DNA and summarize the various methods for mutation screening based on this technique.     

Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis

Mutational analysis of large multiexon genes without prevalent mutations is a laborious undertaking that requires the use of a high-throughput scanning technique. The Human Genome Project has enabled the development of powerful techniques for mutation detection in large multiexon genes. We have transferred heteroduplex analysis (HA) by conformation-sensitive gel electrophoresis of the two major breast cancer (BC) predisposing genes, BRCA1 and BRCA2, to a multicapillary DNA sequencer in order to increase the throughput of this technique. This new method that we have called heteroduplex analysis by capillary array electrophoresis (HA-CAE) is based on the use of multiplex-polymerase chain reaction (PCR), different fluorescent labels and HA in a 16-capillary DNA sequencer. To date, a total of 114 different DNA sequence variants (19 insertions/deletions and 95 single-nucleotide substitutions - SNS) of BRCA1 and BRCA2 from 431 unrelated BC families have been successfully detected by HA-CAE. In addition, we have optimized the multiplex-PCR conditions for the colorectal cancer genes MLH1 and MSH2 in order to analyze them by HA-CAE. Both genes have been amplified in 13 multiplex groups, which contain the 35 exons, and their corresponding flanking intronic sequences. MLH1 and MSH2 have been analyzed in nine hereditary nonpolyposis colorectal cancer patients, and we have found six different DNA changes: one complex deletion/insertion mutation in MLH1 exon 19 and another five SNS. Only the complex mutation and one SNS may be classified as cancer-prone mutations. Our experience has revealed that HA-CAE is a simple, fast, reproducible and sensitive method to scan the sequences of complex genes.     

A Novel Affinity Capillary Electrophoresis for the Detection of Gene Mutation Using Immobilized Oligonucleotides-Polyacrylamide Conjugate

It has been found that small mutations of certain genes are the definitive origin of many heritable disorders and cancers with striking development of recent molecular biology. Such new findings have taken close-up of the importance of gene mutation assays based on the difference of DNA base sequences in diagnostic or medical field Capillary electrophoresis can be a good candidate for an ideal method on such gene analysis, because the methods can be performed with trace amount of samples, high resolution and shorter running time. We have established an effect of oligonucleotide, which was introduced onto capillary inner surface, on the recognition of an overall sequence of sample DNA fragments as an affinity ligand.     

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

PCR/SSCP detects reliably and efficiently DNA sequence variations in large scale screening projects

A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. for clarifying the genetic background of individuals with multifactorial diseases. Here we review our experience with the polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis to identify MPSV in a number of genes thought to be involved in the pathogenesis of multifactorial neurological disorders, including autoimmune diseases like multiple sclerosis (MS) and neurodegenerative disorders like Parkinson s disease (PD). The method is based on the property of the DNA that the electrophoretic mobility of single stranded nucleic acids depends not only on their size but also on their sequence. The target sequences were amplified, digested into fragments ranging from 50-240 base pairs (bp), heat-denatured and analysed on native polyacrylamide (PAA) gels of different composition. The analysis of a great number of different PCR products demonstrates that the detection rate of MPSV depends on the fragment lengths, the temperature during electrophoresis and the composition of the gel. In general, the detection of MPSV is neither influenced by their location within the DNA fragment nor by the type of substitution, i.e., transitions or transversions. The standard PCR/SSCP system described here provides high reliability and detection rates. It allows the efficient analysis of a large number of DNA samples and many different genes.     

DNA typing in hereditary disease.

An increasing number of hereditary diseases are becoming amenable to diagnosis by analysis of DNA as the responsible genes are located and identified. Gel electrophoresis of DNA fragments plays a central role in the diagnosis of hereditary disease. Electrophoretic separation of differently sized fragments enables the characterization or typing of normal variants which are known to be genetically linked to disease genes. For some diseases it is possible to directly detect mutations by DNA electrophoresis. Deletion mutants may be detected by a restriction fragment of altered size or by a failure to amplify a coding region with the polymerase chain reaction. Carriers of small deletions, involving a few base pairs, may be identified by DNA amplification which produces heteroduplexes that show characteristic, anomalous electrophoretic migration. Mutations that alter restriction sites also alter the sizes of restriction fragments. Common disease mutations that alter a single base pair may be detected using a pair of reactions with normal and mutant oligonucleotides under conditions where a perfect match is necessary for hybridization, amplification or ligation. Alternatively a mismatched oligonucleotide primer may be designed to generate a restriction site with either the normal or mutant allele, following DNA amplification. Finally a number of techniques are available that are useful as screening tools for novel mutations.     

The use of denaturing gradient gel electrophoresis to screen for DNA sequence polymorphisms in the human factor VIII gene

We describe the use of denaturing gradient gel electrophoresis to screen for DNA sequence polymorphisms in the human factor VIII gene. DNA fragments that differ in sequence by only a single base pair can be separated on denaturing gradient gels due to changes in their melting behavior. Previous studies have demonstrated the use of denaturing gradient gels to detect sequence changes in human genomic DNA, including mutations in the beta globin gene and polymorphisms on chromosome 20. We have begun to use denaturing gradient gels to look for polymorphisms within the human factor VIII gene. The DNA sequences of seven cloned fragments from introns in the human factor VIII gene were determined and used to predict a melting map for each fragment. The melting behavior of each cloned fragment was evaluated by electrophoresis into denaturing gradient gels. Appropriate fragments were then used as radioactive probes for hybridization to human DNA samples that had been digested with restriction enzymes. Heteroduplexes formed between the probe and genomic DNA samples were electrophoresed into denaturing gradient gels. The final positions of heteroduplex bands were determined by autoradiography. We describe a general approach for using denaturing gradient gel electrophoresis to find DNA polymorphisms, with particular emphasis on the predictive value of DNA sequence data. We compare the efficiency of polymorphism detection by denaturing gradient gel electrophoresis with detection by restriction fragment length polymorphism (RFLP) analysis. The factor VIII gene appears to have a low level of DNA sequence polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single-strand conformation polymorphism method for the large-scale analysis of mutations/polymorphisms using capillary array electrophoresis

We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.     

Optimum sample medium for single-nucleotide polymorphism and mutation detection by capillary electrophoresis

Capillary electrophoresis (CE) is a versatile analytical platform widely used for nucleic acids analysis. Its applications in research and diagnostics include scanning and screening for mutations and polymorphisms by such reliable methods as single-strand conformation polymorphism (SSCP), heteroduplex analysis (HA), and combined SSCP/HA. This study, aimed at the further development of these methods, is focused on detailed sample-media characteristics. Factors affecting single-strand conformer stability and DNA intake efficiency were analyzed. The sample media optimal for efficient mutation or SNP detection were determined, and complex SSCP-CE patterns arising from unpurified PCR products were explained. It turns out, that the nondenaturing aqueous media assure both efficient DNA intake, and single-strand conformers stability required for the SSCP and combined SSCP/HA. The results of this study are applicable to all these areas of biomedical research, in which capillary electrophoresis is used for the characterization of nucleic acids.     

Single-strand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercholesterolemia. The IFCC Scientific Division: Committee on Molecular Biology Techniques.

The identification of the specific mutation causing an inherited disease in a patient is the framework for the development of a rationale for therapy and of DNA-based tests for screening relatives. We present here a review of the single-strand conformational polymorphism (SSCP) method, which allows DNA fragments that have been amplified with specific primers and PCR to be scanned rapidly for any sequence variation. The general principles of the method are described, as are the major factors that must be considered in developing an optimal SSCP strategy, namely length of the PCR fragment and the temperature of the gel run. Options for sample denaturing gel characteristics and detection of DNA fragments are discussed. In addition, several modifications are presented that have been developed for high-throughput mutational analysis. The application of these techniques to screen for mutations in the LDL receptor gene in patients with familial hypercholesterolemia are described.     

Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.     

The potential of electrophoretic mobility shift assays for clinical mutation detection

As the understanding of the links between genetic mutations and diseases continues to grow, there is an increasing need for techniques that can rapidly, inexpensively, and sensitively detect DNA sequence alterations. Typically, such analyses are performed on PCR-amplified gene regions. Automated DNA sequencing by capillary array electrophoresis can be used, but is expensive to apply to large numbers of patient samples and/or large genes, and may not always reveal low-abundance mutations in heterozygous samples. Many different types of genetic differences need to be detected, including single-base substitutions and larger sequence alterations such as insertions, deletions, and inversions. Electrophoretic mobility shift assays seem well suited to this purpose and could be used for the efficient screening of patient samples for sequence alterations, effectively reducing the number of samples that must be subjected to full and careful sequencing. While there is much promise, many of the mobility shift assays presently under development have yet to be demonstrated to have the high sensitivity and specificity of mutation detection required for routine clinical application. Hence, further studies and optimization are required, in particular the application of these methods not only to particular genes but also to large numbers of patient samples in blinded studies aimed at the rigorous determination of sensitivity and specificity. This review examines the state-of-the-art of the most commonly used mobility shift assays for mutation detection, including denaturing gradient gel electrophoresis, TGGE, SSCP, heteroduplex analysis, and denaturing HPLC.     

Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.     

High levels of mitochondrial DNA heteroplasmy in single hair roots: reanalysis and revision.

The present study demonstrates a reinvestigation of the mitochondrial DNA sequence heteroplasmy, which was previously found by the use of nested polymerase chain reaction (PCR) technique in single hairs of 13 individuals. The direct PCR approach was used for the amplification of mitochondrial DNA and a phylogenetic analysis was applied to both data sets for the verification of the authenticity of sequences. The comparative analysis of the sequencing results obtained from the same hair DNA extracts - but using two different techniques - shows that direct mitochondrial DNA amplification results in a considerably lower number of mixed positions. The majority of the confirmed heteroplasmic variants preferentially occurs in mitochondrial DNA hypervariable sites (mutational hotspots). However, the pattern of heteroplasmic mutations observed in four extracts after nested PCR significantly differs from the pattern of natural mutations. Some of these rare polymorphisms should be revised as inconsistent with phylogenetic expectations. The results of the present study contribute to the earlier reports by indicating that phylogenetic analysis is an effective tool in a posteriori quality check of mitochondrial DNA data.     

Twin probes as a novel tool for the detection of single-nucleotide polymorphisms

Single-nucleotide polymorphisms (SNPs) are the most common form of DNA sequence variation. There is a strong interest from both academy and industry to develop rapid, sensitive and cost effective methods for SNP detection. Here we report a novel structural concept for DNA detection based on fluorescence dequenching upon hybridization. The so-called "twin probe" consists of a central fluorene derivative as fluorophore to which two identical oligonucleotides are covalently attached. This probe architecture is applied in homogeneous hybridization assays with subsequent fluorescence spectroscopic analysis. The bioorganic hybrid structure is well suited for sequence specific DNA detection and even SNPs are identified with high efficiency. Additionally, the photophysical properties of the twin probe were investigated. The covalent attachment of two single stranded oligonucleotides leads to strong quenching of the central fluorescence dye induced by the nucleobases. The twin probe is characterized by supramolecular aggregate formation accompanied by red-shifted emission and broad fluorescence spectra.     

High-throughput screening of genetic mutations/polymorphisms by capillary electrophoresis

Genetic mutations/polymorphisms analyses play a great role in genetic and medical research, and clinical diagnosis. Most conventional methods for genetic assay are based on slab gel electrophoresis that is both labor-intensive and time-consuming. Recently, capillary electrophoresis (CE) has been used for genetic analysis instead of conventional slab gel electrophoresis. This technique can be automated and is characterized by short analysis time, small sample and reagents requirements, and high separation efficiency. CE has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes, and has shown a great potential for genetic mutation/polymorphism screening of large numbers of clinical samples. In this article, an overview of the fundamental aspects of mutation/polymorphism assay methods in combination with CE is given and some key applications are summarized.     

Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Detection of point mutations in driver genes is of great significance for the early diagnosis, treatment, and prognostic evaluation of cancer. However, current detection methods do not offer versatility, specificity,and rapid performance simultaneously. Thus, multiple mutation detection processes are necessary, which results in long processing times and high costs. In this study, we developed a thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detec...