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.     

Efficient mutation detection in MEN1 gene using a combination of single-strand conformation polymorphism (MDGA) and heteroduplex analysis

For facilitated genotypic analysis of multiple endocrine neoplasia type 1 (MEN1), a familial syndrome associated with tumors of the parathyroid and neuroendocrine tissues, we developed two screening methods, heteroduplex mutation assay (HMA) and mutation detection gel analysis (MDGA), both based on electrophoretic discrimination of polymerase chain reaction (PCR) products, to detect the mutations. Forty-three genomic DNA samples were used for the evaluation of these techniques. The whole coding region of MEN1 was PCR-amplified with fluorescent primers and then denatured/renatured before electrophoresis on an automated sequencer. 100% of the mutations were detected, subsequently confirmed and identified by sequencing. "Negative" samples were used to evaluate the specificity and reproducibility of the two techniques. The combination of the two methods allows high throughput cost-effective mutation screening which is less laborious than systematic sequencing of the whole coding region of MEN1. Together, these methods provide an efficient screen for MEN1 mutations.     

Mutations in mitochondrial-encoded cytochrome c oxidase subunits I, II, and III genes detected in Alzheimer's disease using single-strand conformation polymorphism

A "mitochondrial hypothesis" of late onset Alzheimer's disease (AD) has been proposed. Biochemical studies indicate that there is a significant decrease in cytochrome oxidase (CO) activity as well as perturbed CO I and CO III mRNA levels in platelets and brain tissue from Alzheimer's patients. Using the electrophoretic mutation detection technique SSCP and DNA sequencing, we have identified 20 point mutations in the mitochondrial-encoded CO subunits (CO I, II, and III) in AD and age-matched control brain samples. Eight of the mutations are new variants of the mitochondrial genome. The efficiency of SSCP in detecting mutations in the CO subunits was estimated to be 80% when compared to dideoxy sequencing. One of the mutations (at position 9,861) results in a phenylalanine-->leucine substitution at a highly conserved residue in CO III. CO activity was reduced by an average of 35% in all AD brains compared to age-matched control samples, which agrees with previous reports. CO activity in one of the AD brain samples carrying the 9,861 mutation decreased by 80% relative to control brain samples, suggesting that the phenotypic expression of this mutation may result in reduced CO activity and compromised mitochondrial function.     

Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays

Nucleotides with a dye attached to the terminal phosphate with four or more phosphates (tetra- or pentaphosphates) are superior substrates than the corresponding triphosphates for DNA and RNA polymerases. When fluorogenic dyes are directly attached to the terminal phosphate, they can be released by the action of polymerase and alkaline phosphatase. The released dye changes color and fluorescence properties. The fluorescent signal can also be amplified by using multiple labeled nucleotides to detect small amounts of template. We have explored the utility of these nucleotides in a variety of applications including homogeneous SNP detection methods, DNA sequencing, and quantitation of PCR and RCA.     

Phantom mutation hotspots in human mitochondrial DNA

Phantom mutations are systematic artifacts generated in the course of the sequencing process. Contra common belief these artificial mutations are nearly ubiquitous in sequencing results, albeit at frequencies that may vary dramatically. The amount of artifacts depends not only on the sort of automated sequencer and sequencing chemistry employed, but also on other lab-specific factors. An experimental study executed on four samples under various combinations of sequencing conditions revealed a number of phantom mutations occurring at the same sites of mitochondrial DNA (mtDNA) repeatedly. To confirm these and identify further hotspots for artifacts, > 5000 mtDNA electropherograms were screened for artificial patterns. Further, > 30 000 published hypervariable segment I sequences were compared at potential hotspots for phantom mutations, especially for variation at positions 16085 and 16197. Resequencing of several samples confirmed the artificial nature of these and other polymorphisms in the original publications. Single-strand sequencing, as typically executed in medical and anthropological studies, is thus highly vulnerable to this kind of artifacts. In particular, phantom mutation hotspots could easily lead to misidentification of somatic mutations and to misinterpretations in all kinds of clinical mtDNA studies.     

Automated detection of differently expressed fragments in mRNA differential display

We present a novel method for the automated detection of fragments showing dissimilar expression in mRNA differential display. The analysis is based on aligning the numerical electrophoretic lane data in respect of a given distance function defined on a set of fragments, or signal peaks in general. We presume that significant dissimilarities between peaks result in extreme score values computed for aligned peak pairs. Whereas in sequence comparison, an overall sequence similarity score is conventionally used, the current method defines a special dissimilarity score for searching the peak pairs showing the largest relative differences between the lanes. The output of the analysis is a highly reduced list of peak pairs, along with a set of associated features extracted from the lanes. Only the peaks of this list need to be visually confirmed instead of the vast amount of peaks in the original electrophoretic results. The results obtained by the algorithm correlate well with results of visual evaluation of the same electropherograms. The current algorithm may be applied to the study of complex expression patterns in multiple lanes and, in general, to automated recognition of variously defined patterns of quantitative electrophoretic data.     

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.     

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.     

BRAF mutation detection and identification by cycling temperature capillary electrophoresis

BRAF mutations are found in many human tumors, namely melanomas ( approximately 70%) and colon carcinomas ( approximately 15%). This paper presents a method for identification of exon 15 BRAF mutations by denaturant capillary electrophoresis (CE), an analysis method that is sensitive, cost-effective (involving only polymerase chain reaction (PCR) and electrophoresis) and capable of high-throughput screening. In total, we found 21 (70%) out of 30 melanoma cell lines with BRAF mutations in exon 15: two of which were the p.Val600Asp (c.1799-800TG>AT) mutation, one cell line contained the p.Val600Arg (c.1798-99GT>AG) mutation, and 18 cell lines contained the p.Val600Glu (c.1799T>A) mutation. Of the nine cell lines that did not contain a BRAF mutation, five contained an NRAS mutation at exon 2, and no mutations were detected in NRAS exon 1. There was no overlap of NRAS and BRAF mutations in the same cell line. In addition, we looked at 221 colon biopsy samples and identified one further BRAF mutation, the p.Asp594Gly (c.1781A>G) mutation, in seven samples. The p.Val600Glu mutation was identified in 11 of the colon biopsy samples. Using the four mutations of BRAF exon 15, we then constructed a denaturing CE standard capable of distinguishing between each of the mutations; therefore, sequencing does not need to be performed to confirm the mutation. In conclusion, this sensitive, cost-effective mutation assay for BRAF (and RAS) will provide the opportunity to detect and determine mutations without the need to purify samples for sequencing. Future large-scale studies will provide the clinical usefulness of such mutations.     

Electrochemical Detection of FAM134B Mutations in Oesophageal Cancer Based on DNA‐Gold Affinity Interactions

Inexpensive, simple and rapid DNA sensors capable of accurate and sensitive detection of cancer specific point mutations in DNA biomarkers are crucial for the routine screening of genetic mutations in cancer. Conventional approaches based on sequencing, mass spectroscopy, and fluorescence are highly effective, but they are tedious, slow and require labels and expensive equipment. Recent electrochemistry based approaches mostly rely on conventional DNA biosensing using recognition and transduction layers, and hence limited by the complicated steps of sensor fabrication associated with surface cleaning, self‐assembled monolayer formation, and target hybridization. Herein we report a relatively simple and inexpensive method for detecting point mutation in cancer by using the direct adsorption of purified DNA sequences onto an unmodified gold surface. The method relies on the base dependent affinity interaction of DNA with gold. Since the affinity interaction (adsorption) trend of DNA bases follows as adenine (A) > cytosine (C) > guanine (G)> thymine (T), two DNA sequences with different DNA base compositions (i. e., amplified mutated sequences will be distinctly different than its original sequence) will have different adsorption affinity towards gold. The amount of mutation sites on a DNA sequence is quantified by monitoring the electrochemical current as a function of the relative adsorption level of DNA samples onto a bare gold electrode. This method can successfully distinguish single point mutation in DNA from oesophageal cancer. We demonstrated the clinical utility of this approach by detecting different levels of mutations in tissue samples (n=9) taken from oesophageal cancer patients. Finally, the method was validated with High Resolution Melt (HRM) curve analysis and Sanger Sequencing.     

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.     

A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3′ (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the “wrong” genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3′ exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3′-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3′-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.     

Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique is developed for the detection of point mutations in DNA samples, and is very useful in the research of tumors. The traditional SSCP was carried out with slab gel electrophoresis (SGE), but this is time-consuming and labor-intensive, particularly for clinical diagnoses. We have developed a capillary electrophoresis (CE) method for SSCP detection with a linear polyacrylamide gel solution as the sieving matrix. Twenty colon tumor samples were detected with SSCP-CE and the point mutation in exon 7 of the p53 gene was found in six of the samples. Based on the sequencing results, the accuracy of SSCP-CE was better than that of SSCP-SGE. We hope this rapid and convenient method could be applied in the clinical diagnosis of tumors soon.     

A statistical model for unwarping of 1-D electrophoresis gels

A statistical model is proposed which relates density profiles in 1-D electrophoresis gels, such as those produced by pulsed-field gel electrophoresis (PFGE), to databases of profiles of known genotypes. The warp in each gel lane is described by a trend that is linear in its parameters plus a first-order autoregressive process, and density differences are modelled by a mixture of two normal distributions. Maximum likelihood estimates are computed efficiently by a recursive algorithm that alternates between dynamic time warping to align individual lanes and generalised-least-squares regression to ensure that the warp is smooth between lanes. The method, illustrated using PFGE of Escherichia coli O157 strains, automatically unwarps and classifies gel lanes, and facilitates manual identification of new genotypes.     

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.     

A general approach to the analysis of errors and failure modes in the base-calling function in automated fluorescent DNA sequencing

We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.     

Mobility and activation energy of single-stranded DNA in denaturing cross-linked polyacrylamide slab gels

An original apparatus based on laser-induced fluorescence detection is presented. One lane migration combined to four equidistant detection points allows the study of the dynamics of DNA bands during electrophoresis. We focus this article on the study of the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T, 5% C and 4.3% T, 5% C cross-linked polyacrylamide gels at an electric field of 45 V/cm [T=(g acrylamide+g N,N'-methylenebisacrylamide)/100 ml solution; C=g N,N'-methylenebisacrylamide/% T]. Activation energy has been investigated under these experimental conditions with a temperature varying from 25 to 50 degrees C. The activation energy for migration through the cross-linked polyacrylamide gel decreases with fragment length under our experimental conditions and it varies along the migration.     

Compensation for mobility inequalities between lanes computed from band signals in on-line fluorescence DNA sequencing.

Among on-line fluorescence DNA sequencing systems, the four-lane method exhibits the potential for reporting an erroneous sequence due to nonuniform mobility of the DNA fragments migrating among the four lanes. This error is manifest in phenomenon commonly called smiling. This paper presents a computational algorithm which compensates for the mobility inequalities between lanes using signal data obtained from the shorter DNA fragments forming the faster migrating bands. The program mainly consists of two routines: (i) calculation of calibration coefficients (mobility ratios between lanes), and (ii) examination of the coefficients by applying them to a later domain of the same signals. Both routines are connected with several feed-back branches for recalculation. Homology analysis of final sequences has shown that the accuracy rate is maximized with this algorithm and any ambiguous result can be assigned to the residual error inherent in the band identification method used.     

Analysis of electrophoretic patterns of arbitrarily primed PCR profiling

We present a mathematical algorithm for the analysis of electrophoretic patterns resulting from arbitrarily primed PCR profiling. The algorithm is based on the established mathematical procedures applied to the analysis of digital images of gel patterns. The algorithm includes (a) transformation of the image into a matrix form, (b) identification of every electrophoretic lane as a set of matrix columns that are further mathematically processed, (c) averaging of matrix columns corresponding to electrophoretic lanes that define lane representatives, (d) elimination of "smiling" bands, (e) solving the problem of a lane offset, and (f) removal of the background. Representation of individual electrophoretic lanes in the form of functions allows interlane comparisons and further mathematical analysis. Direct comparison of selected lanes was obtained by employing correlation analysis. Gel images were those obtained after arbitrarily primed PCR analysis of DNA that underwent damage induced by gamma radiation from a (60)Co source. The applied method proved to be useful for elimination of subjectivity of visual inspection. It offers the possibility to avoid overlooking important differences in case of suboptimal electrophoretic resolution. In addition, higher precision is achieved in the assessment of quantitative differences due to better insight into experimental artifacts. These simple mathematical methods offer an open-type algorithm, i.e., this algorithm enables easy implementation of different parameters that may be useful for other analytical needs.