Jespersgaard, C. et al. Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics. Electrophoresis 27, 3816-3822

Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark.
Electrophoresis (Impact Factor: 3.03). 10/2006; 27(19):3816-22. DOI: 10.1002/elps.200600095
Source: PubMed


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
10 Reads
  • Source
    • "We used capillary electrophoresissingle strand conformation polymorphism (CE-SSCP) analysis to distinguish between mild and severe CTV isolates (Raspagliesi et al., 2011a; Licciardello et al., 2012). This approach combines the potential of SSCP analysis with the advantages of automation and the standardization of capillary electrophoresis, which provides greater resolving power and increased sensitivity and also enables analyzing multiple samples simultaneously (Jespersgaard et al., 2006). Combining sequential analysis by immunoenzymatic tests and CE-SSCP also ensures a safe working environment, even with isolates from foreign countries (Licciardello et al., 2012). "
    [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
  • Source
    • "Capillary electrophoresis SSCP (CE-SSCP), which has been used previously to detect mutations (Larsen et al., 2007), construct profiles of bacterial communities (Sen et al., 2008) and categorize isolates of influenza virus (Quinto and Wang, 2004), combines the potential of single-strand conformation analysis with the automation and standardization of electrophoresis run conditions. In this manner, CE-SSCP provides greater resolving power and increased sensitivity and also allows the simultaneous analysis of multiple samples (up to 96 in a single run) with a throughput of 100 samples per day, all without intensive labor (Jespersgaard et al., 2006). Recently, CE-SSCP has also been used to study plant-pathogenic viruses, allowing the quick characterization of CTV isolates (Raspagliesi et al., 2011) and the analysis of the spatial distribution of a Plum pox virus population (Dallot et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Citrus tristeza virus (CTV) is the causal agent of tristeza disease, which is one of the most devastating diseases of citrus crops worldwide. This paper describes a method for the rapid detection and genotyping of naturally spreading CTV isolates. This method uses ELISA or dot-blot immunological tests to detect trees infected with CTV. The reaction wells or membrane spots for which there is a positive reaction are sequentially treated by (i) washing and elution of viral RNA from the trapped samples, (ii) one-step synthesis of cDNA and PCR and (iii) automated fluorescence-based capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis of amplification products. Comparative CE-SSCP results are presented for CTV RNA extracted directly from infected leaves and ELISA plates or from membranes. In the analyses of all of these RNA samples, the p18, p27 and p23 CTV genes were targeted for amplification. Specific profiles of forward and reverse strands were obtained from a group of eight CTV isolates collected in Sicily, each with distinct biological characteristics, which were analyzed using the conventional two-step procedure (immunological detection followed by CE-SSCP molecular characterization after RNA isolation) or in a continuous process of ELISA/CE-SSCP or dot-blot/CE-SSCP starting from infected plant material. The combined method is simple, highly sensitive and reproducible, thus allowing the processing of numerous field samples for a variety of epidemiological needs. The sequential processing of an ELISA or dot-blot/ELISA followed by CE-SSCP is expected to allow the rapid detection of recent CTV infections along with the simultaneous characterization of the genetic diversity and structure of the population of newly invading CTV.
    Journal of virological methods 01/2012; 181(2):139-47. DOI:10.1016/j.jviromet.2012.01.015 · 1.78 Impact Factor
  • Source
    • "Effective methods for mutation screening in the coding region of SCN5A using CAE-SSCP [Hofman-Bang et al., 2006], DHPLC [Lai et al., 2005; Ning et al., 2003], or Melting Curve Analysis [Millat et al., 2009] have been described. Irrespective of the method used, it is important that the sensitivity and specificity of the method used is validated in the implementing laboratory [Jespersgaard et al., 2006]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Brugada syndrome (BrS) is a condition characterized by a distinct ST-segment elevation in the right precordial leads of the electrocardiogram and, clinically, by an increased risk of cardiac arrhythmia and sudden death. The condition predominantly exhibits an autosomal dominant pattern of inheritance with an average prevalence of 5:10,000 worldwide. Currently, more than 100 mutations in seven genes have been associated with BrS. Loss-of-function mutations in SCN5A, which encodes the alpha-subunit of the Na(v)1.5 sodium ion channel conducting the depolarizing I(Na) current, causes 15-20% of BrS cases. A few mutations have been described in GPD1L, which encodes glycerol-3-phosphate dehydrogenase-1 like protein; CACNA1C, which encodes the alpha-subunit of the Ca(v)1.2 ion channel conducting the depolarizing I(L,Ca) current; CACNB2, which encodes the stimulating beta2-subunit of the Ca(v)1.2 ion channel; SCN1B and SCN3B, which, in the heart, encodes beta-subunits of the Na(v)1.5 sodium ion channel, and KCNE3, which encodes the ancillary inhibitory beta-subunit of several potassium channels including the Kv4.3 ion channel conducting the repolarizing potassium I(to) current. BrS exhibits variable expressivity, reduced penetrance, and "mixed phenotypes," where families contain members with BrS as well as long QT syndrome, atrial fibrillation, short QT syndrome, conduction disease, or structural heart disease, have also been described.
    Human Mutation 07/2009; 30(9):1256-66. DOI:10.1002/humu.21066 · 5.14 Impact Factor
Show more