Scanning for DNA Variants by Denaturant Capillary Electrophoresis

Ekstrøm, Per Olaf

doi:10.1007/978-1-59745-188-8_2

Cited by 3 publications

(1 citation statement)

References 40 publications

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“…CGE has traditionally been an inherent part of molecular genetic testing protocols applied for DNA sequencing, DNA fragment analysis, and detection of DNA variations (mutations and polymorphisms) [16][17][18]. Over the past years, a number of publications have described a special experimental setting in which half-melted mutant homo-and heteroduplex forms are resolved by CE running at partial denaturing conditions [19][20][21]. Because of the low demand on amounts of input DNA and high sensitivity allowing identification of small fractions of mutated DNA in the sample, this approach has found its main use in detection of somatic DNA mutations in cancerous tissue [22][23][24][25][26].…”

Section: Introductionmentioning

confidence: 99%

Application of denaturing capillary electrophoresis for the detection of prognostic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes in brain tumors

Benešová

Belšánová

Kramář

et al. 2018

J of Separation Science

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Malignant transformation in gliomas is frequently supplemented by somatic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes. It has recently emerged that mutations in these genes are associated with prolonged survival and should be used as prognostic factor in management of brain cancer patients. There are several approaches in use for the detection of isocitrate dehydrogenase 1 and 2 mutations; however, these often exhibit shortcomings such as convoluted protocols with long processing time, complex (and costly) dedicated fluorescent probes, and/or demand on amounts of input DNA. Therefore, a simple and rapid method would be highly desired. Here, we present development and validation of simple and reliable isocitrate dehydrogenase 1 and 2 mutation detection assay using denaturing capillary electrophoresis. The detection sensitivity in terms of the limiting mutated allele fraction detectable estimated from a series of dilution runs was 2.9%. The method was validated by comparing to results obtained by a widely accepted detection technique, the multiplex ligation-dependent probe amplification, on a set of 85 brain tumors. The concordance of both methods was 100%, but denaturing capillary electrophoresis assay required fivefold lower input of DNA (1 versus 5 μL of DNA at concentrations typically between 10 and 30 ng/μL).

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Section: Introductionmentioning

confidence: 99%

Application of denaturing capillary electrophoresis for the detection of prognostic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes in brain tumors

Benešová

Belšánová

Kramář

et al. 2018

J of Separation Science

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Denaturing capillary electrophoresis for automated detection of L858R mutation in exon 21 of the epidermal growth factor receptor gene in prediction of the outcome of lung cancer therapy

Benešová

Pešek²,

Belšánová

et al. 2010

J of Separation Science

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The presence of activating mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene has been attributed to a positive response to biological therapy of lung cancer by small-molecular tyrosine kinase inhibitors, gefitinib and erlotinib. Among the two most significant mutation types are deletions in exon 19 and a single point substitution in exon 21 (termed L858R). The exon 19 deletions can readily be examined by fragment analysis, due to the characteristic length difference between the normal and mutated PCR product. Analysis of the L858R point mutation, however, presents a greater challenge. The current paper is aimed at developing a sensitive, yet simple, low-cost mutation detection assay directed at the L858R mutation using a method based on CE of heteroduplexes under partial denaturing conditions. We perform optimization of separation conditions on different commercial instruments including ones equipped with 8, 16 and 96 capillaries. We present normalized migration reproducibility in the range from 1 (8 and 16) to 5% (96) RSD. A reliable distinction of the R836R silent polymorphism from a potential presence of the L858R mutation is also demonstrated. In its implementation, the presented assay is just another application running on a conventional CE platform without the need of dedicated instrumentation.

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Genetic variation in FOXP2 alters grey matter concentrations in schizophrenia patients

Španiel

Horáček

Tintěra

et al. 2011

Neuroscience Letters

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Scanning for DNA Variants by Denaturant Capillary Electrophoresis

Cited by 3 publications

References 40 publications

Application of denaturing capillary electrophoresis for the detection of prognostic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes in brain tumors

Application of denaturing capillary electrophoresis for the detection of prognostic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes in brain tumors

Denaturing capillary electrophoresis for automated detection of L858R mutation in exon 21 of the epidermal growth factor receptor gene in prediction of the outcome of lung cancer therapy

Genetic variation in FOXP2 alters grey matter concentrations in schizophrenia patients

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