Article

Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics

Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark.
Electrophoresis (Impact Factor: 3.16). 10/2006; 27(19):3816-22. DOI: 10.1002/elps.200600095
Source: PubMed

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.

1 Follower
 · 
110 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA methylation is a complex event in epigenetic studies because of both the large CpG islands present upstream of the promoter region and the different distribution of DNA methylation despite similar methylation levels. For this reason, we proposed a fast, cost-effective method for the screening of DNA methylation based on single-strand conformation polymorphism (SSCP) and CE-LIF. In this study, the PCR products that were amplified from bisulfite-treated genomic DNA were denatured at 94°C, followed by immediate chilling in ice water to form the single-strand DNA (ssDNA). The ssDNA were separated by 1.5% poly(ethylene oxide) (Mavg 8,000,000 Da) in the presence of EOF according to the different conformations represented by their unique methylation states. This result demonstrated that four hepatocellular carcinoma cell lines represented a different heterogeneity of DNA methylation and could be distinguished by SSCP-CE. The results obtained from SSCP-CE also corresponded with those obtained from combined bisulfide restriction analysis and methylation-sensitive high-resolution melting analysis. Therefore, the proposed SSCP-CE method may potentially be used for rapid screening for determination of the heterogeneity of DNA methylation in further epigenetic studies and clinical diagnosis. This article is protected by copyright. All rights reserved.
    Electrophoresis 08/2014; 35:2378-2385. DOI:10.1002/elps.201300502 · 3.16 Impact Factor
  • [Show abstract] [Hide abstract]
    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. DOI:10.1002/elps.201300078 · 3.16 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The genetic structure of the Citrus tristeza virus (CTV) population in the Hunan Province, China, was studied by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP), multiple molecular markers (MMM), and bioindexing. A total of 17 samples collected in Yizhang and Yong Xing counties were tested by tissue print-ELISA, and 10 were found CTV infected. CE-SSCP analysis showed different profiles some of them indicating composite patterns. MMM analysis revealed the presence of the VT and T3 genotypes, either individually or in combination. One more isolate from Chenzou (HU-PSTS) showing the most complex CE-SSCP profile and a T36 + VT + T30 genotype was further investigated by graft inoculation on indicator plants and phylogenetic analysis of the p25 gene. Bioindexing revealed a very severe seedling yellows reaction and an unusual stem pitting on sour orange. Three p25 clones obtained from this isolate, showed different CE-SSCP profiles and were phylogenetically close to recombinant Hawaiian and Indian strains. INTRODUCTION With more than 320,000 ha of citrus trees, the Hunan Province provides 15% of the total production in China and is one of the most important producers in the world. The use of tolerant rootstocks has long protected the citrus industry from the devastating effects of tristeza decline caused by the Citrus tristeza virus (CTV). However, currently stem pitting (SP) disease, also produced by CTV (Moreno et al., 2008), is causing significant damage to the citrus production. Isolates inducing seedling yellows (SY), the third syndrome incited by CTV, have also been recorded (Zhou et al., 1996). CTV has been reported in Hunan and other citrus areas in China as a result of surveys based on ELISA (Ke et al., 1984). Our group surveyed several citrus-growing areas in Hunan Province and tested over 100 samples for CTV infection. Biological indexing, ELISA and PCR showed that 92% of the samples were CTV infected and that 4 to 6% caused SP (Rizza et al., 2010). Zhou et al. (1996) also found differences in nine CTV isolates by biological indexing, with some inducing severe SP and SY. However, data on the molecular characteristics of Chinese CTV isolates are scarce. Jiang et al. (2008) studied the association between symptoms and CP gene sequences and found two mild strains showing a high identity with the isolates T30 (Florida) and T385 (Spain). Their results suggest that restriction fragment length polymorphism (RFLP) analysis with HinfI may be able to discriminate between mild and severe strains in China. Yi et al. (2007) analyzed CTV isolates from wild-type citrus to evaluate genetic variations and gain insight into the evolution of CTV. Our group also ran a preliminary study to discriminate between mild and severe strains based on specific amino acids of the p23 protein (Song et al., 2006; Dai et al., 2010). Perhaps the most complete information on the structure of the virus population was reported by Hilf et al. (2005) on 22 Chinese isolates, for which multiple molecular marker (MMM) analysis was done.
    XII International Citrus Congress, Valencia, Spain; 01/2015