Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics.
ABSTRACT Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.
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ABSTRACT: Adult tapeworms of the genus Echinococcus (family Taeniidae) occur in the small intestines of carnivorous definitive hosts and are transmitted to particular intermediate mammalian hosts, in which they develop as fluid-filled larvae (cysts) in internal organs (usually lung and liver), causing the disease echinococcosis. Echinococcus species are of major medical importance and also cause losses to the meat and livestock industries, mainly due to the condemnation of infected offal. Decisions regarding the treatment and control of echinococcosis rely on the accurate identification of species and population variants (strains). Conventional, phenetic methods for specific identification have some significant limitations. Despite advances in the development of molecular tools, there has been limited application of mutation scanning methods to species of Echinococcus. Here, we briefly review key genetic markers used for the identification of Echinococcus species and techniques for the analysis of genetic variation within and among populations, and the diagnosis of echinococcosis. We also discuss the benefits of utilizing mutation scanning approaches to elucidate the population genetics and epidemiology of Echinococcus species. These benefits are likely to become more evident following the complete characterization of the genomes of E. granulosus and E. multilocularis.Electrophoresis 07/2013; 34(13):1852-62. · 3.26 Impact Factor
Conference Paper: Plastic network for predicting the Mackey-Glass time series[Show abstract] [Hide abstract]
ABSTRACT: A novel plastic network is introduced as a tool for predicting chaotic time series. When the goal is prediction accuracy for chaotic time series, local-in-time and local-in-state-space plastic networks can outperform the traditional global methods. The key ingredient of a plastic network is a model selection criterion that allows it to self organize by choosing among a collection of candidate models. Among the advantages of the plastic network for the prediction of (chaotic) time series are the simplicity of the models used, accuracy, relatively small data requirement, online usage, and ease of understanding of the algorithms. When reporting prediction results on chaotic time series, a careful analysis of the data is recommended. Specifically for the Mackey-Glass time series, the authors find that different forward lead size can result in different prediction accuracyNeural Networks, 1992. IJCNN., International Joint Conference on; 07/1992
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ABSTRACT: 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.Electrophoresis 08/2002; 23(14):2259-66. · 3.26 Impact Factor