Theory of band broadening during cycling temperature capillary electrophoresis

Laachi, Nabil; Dorfman, Kevin D.

doi:10.1002/elps.200600565

Cited by 6 publications

(7 citation statements)

References 18 publications

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“…Temperature oscillations may also be used to separate homoduplex and heteroduplex DNA. It was predicted that movement of DNA through temperature zones causes alternate annealing and melting of DNA, ultimately affecting its observed electrophoretic mobility (94). The prediction stated that DNA with and without a point mutation will have a different lag in annealing that would result in different mean velocities.…”

Section: Nucleic Acid Analysis (1) Fundamental Studiesmentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

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Science reports ∼4800 hits on capillary electrophoresis (CE) including 410 reviews. Because of the prominence of CE techniques in bioanalysis, we decided make them the focus of this review and selected 200 hundred papers representing advances in this field. The selected papers cover advances in CE theory, instrumentation, and methodologies that are specific to various analytes of biological origin or relevance. The group of analytes includes nucleic acids, proteins and peptides, carbohydrates, lipids, single cells, and bioparticles. In addition, we have included advances in the use of CE to define functional assays or to investigate biomolecular interactions. The use of microfabricated devices for CE analysis was not included because this is already covered in other review. Technique Developments Separation SchemesDetermining the velocity of the electroosmotic flow (EOF) and how it changes during an electrophoretic separation is still an important research topic. A simple method for EOF measurements using so-called thermal marks was reported (1). Here, a tungsten filament caused punctual heating at the capillary wall and caused a perturbation in the electrolyte concentration. A sequence of these "thermal marks" then migrated with the EOF until each mark reached and was detected by a conductivity detector. The feasibility of using thermal marks as internal EOF standards in different separation systems was thereby demonstrated.Isoelectric focusing separates amphoteric analytes such as proteins or peptides by the differences in their isoelectric points. Most of the reports on capillary isoelectric focusing (cIEF) describe an initial focusing phase after which the focused zones are mobilized and detected. A dynamic cIEF method for protein analysis was reported (2). This technique made it possible to control each protein's position and focused width by moving the pH gradient within the capillary through manipulation of the electric fields. An important advantage of this approach is the capability of collecting focused analytes from the central section, suggesting that there may be great potential for introducing selectively focused proteins to a second separation dimension such as LC or CE.Micellar electrokinetic capillary chromatography (MEKC) is typically incompatible with electrospray mass spectrometry (ESI-MS) because the nonvolatile surfactants in the micellar phase result in complicated adduct formation and loss of sensitivity during the electrospray process and because the presence of the organic solvent needed for electrospray may cause instability in the micellar phase. These drawbacks were overcome by using synthetic polymeric surfactants that can work as a pseudostationary phase and provide stable electrospray (3). The polymeric surfactant was made by polymerizing three amino acidderived (L-leucinol, L-isoleucinol, L-valinol) sulfated chiral surfactants. These polymeric

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Section: Nucleic Acid Analysis (1) Fundamental Studiesmentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

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“…Indeed, the temperature uniformity of the 96‐capillary instrument was partially due to the addition of the sandwich thermostat. The variation across larger capillary arrays could further be improved if cycling temperature gradient is applied as recently demonstrated by theoretical modeling 32 as well as by previous experimental results 33.…”

Section: Discussionmentioning

confidence: 85%

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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“…While the basic CTCE method was published in 2002, the separation principles have not been fully elucidated. One attempt to describe the theoretical principle of the separation has been made in reference [], but the authors lacked the experimental data to verify the model. In this report, we provide data that can be used to predict the separation of alleles based on theoretical melting differences, temperature ramp rate within the capillary chamber, and fragment velocity in the two different states—annealed (v a ) and partially melted (v m )—under defined electrophoretic conditions.…”

Section: Resultsmentioning

confidence: 99%

“…It has been postulated that the enforced melting and reannealing should cause band broadening []. To test the possible band‐broadening effect of temperature cycling, fragments were analyzed at low constant temperature (35°C) to ensure that the DNA was in an annealed double stranded form, and at cycling temperature ([46–43°C] × 20) around the theoretical melting temperature.…”

Section: Resultsmentioning

confidence: 99%

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Separation principles of cycling temperature capillary electrophoresis

2012

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High throughput means to detect and quantify low-frequency mutations (<10(-2) ) in the DNA-coding sequences of human tissues and pathological lesions are required to discover the kinds, numbers, and rates of genetic mutations that (i) confer inherited risk for disease or (ii) arise in somatic tissues as events required for clonal diseases such as cancers and atherosclerotic plaque.While throughput of linear DNA sequencing methods has increased dramatically, such methods are limited by high error rates (>10(-3) ) rendering them unsuitable for the detection of low-frequency risk-conferring mutations among the many neutral mutations carried in the general population or formed in tissue growth and development. In contrast, constant denaturing capillary electrophoresis (CDCE), coupled with high-fidelity PCR, achieved a point mutation detection limit of <10(-5) in exon-sized sequences from human tissue or pooled blood samples. However, increasing CDCE throughput proved difficult due to the need for precise temperature control and the time-consuming optimization steps for each DNA sequence probed. Both of these problems have been solved by the method of cycling temperature capillary electrophoresis (CTCE). The data presented here provide a deeper understanding of the separation principles involved in CTCE and address several elements of a previously presented two-state transport model.

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Theory of band broadening during cycling temperature capillary electrophoresis

Cited by 6 publications

References 18 publications

Capillary Electrophoresis in Bioanalysis

Capillary Electrophoresis in Bioanalysis

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

Separation principles of cycling temperature capillary electrophoresis

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