Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

Heiger, David N.; Cohen, Aharon S.; Karger, Barry L.

doi:10.1016/s0021-9673(01)90202-x

Cited by 399 publications

(149 citation statements)

References 30 publications

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“…There are many factors that govern the electrophoretic separation quality. Because such a topic has been detailed by others, [14][15][16][17][18][19][20][21][22]25,26 only a brief description is given here.…”

Section: Optimization Of Ce Methodsmentioning

confidence: 99%

“…However, difficulties in preparing a capillary gel column, such as the formation of a bubble and the low duration of high voltage, have bottlenecked its further development. Recently, linear polymers have been popularly used in CE as the sieving matrix instead of the crosslinked gel, in terms of non-gel sieving or an entangled polymersolution CE, 13,14 due to its facilitation 15 of high separation speed, long read length and replaceable operation compared to CGE. Many polymers have proved to be useful, including linear polyacrylamide and its derivatives, 14,16,17 cellulose and its derivatives, 18,19 polyethene oxide 20,21 and agarose 22 etc.…”

Section: Introductionmentioning

confidence: 99%

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Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

Liu

Xie

2001

ANAL. SCI.

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It was demonstrated that a capillary electrophoresis (CE) method with a non-gel sieving solution has been developed to identify the orientation of DNA fragments in recombinant plasmids in molecular biology. The influences of the concentration of sieving polymer HEC, the applied electric field strength and sampling on CE separation were analyzed concerning the optimization of separation. YO-PRO-1 was used as a DNA intercalating reagent to facilitate fluorescence detection. Under the chosen conditions (buffer, 1 × TBE containing 1 µM YO-PRO-1 and 1.2% HEC; applied electric field strength, 200 V/cm; electrokinetic sampling: time, 5 s; voltage, -6 kV), three DNA markers (φ174/HaeIII, pBR322/HaeIII and λDNA/HindIII) were tested for further evaluating the relationship between the DNA size and the mobility. The established CE method conjugated with the enzymatic approach was successfully applied to identifying the DNA orientation of recombinant plasmid in transgene operations of a newly cloned gene from Arabidopsis Thaliana.

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Section: Optimization Of Ce Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

Liu

Xie

2001

ANAL. SCI.

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“…From Equation (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) the stress is found to be a = 202 MPa. Since this is well below the yield strength of the fixture and given the use of worst case conditions, it can be assumed that the fixture will not yield due to the thermal expansion of the gradient plate.…”

Section: -E-i 3-e-imentioning

confidence: 99%

DNA mutation detection via fluorescence imaging in a spatial thermal gradient, capillary electrophoresis system

Crane

Hogan

Lerman

et al. 2001

Review of Scientific Instruments

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To improve the speed and efficiency of genetic screening, we are developing an instrument to detect mutations via a high throughput, automated system. Detection is based on changes in the melting temperature induced by single point mutations as in Denaturing Gradient Gel Electrophoresis (DGGE). This instrument measures the migrating position of test and wild type molecules in a spatial thermal gradient during capillary electrophoresis. In this thesis, the concept is described. A thorough design process is conducted focusing on controlling thermal expansion, creating a stable, predictable temperature gradient, and optimizing software and hardware. Finally, the concept is proved in a series of experiments successfully identifying the presence of a mutation in the test sample.

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“…Traditionally, DNA has been analyzed almost exclusively by slab-gel electrophoresis (SGE) and this is still an important tool in biochemistry and molecular biology laboratories all over the world. However, since its earlier stages CE has shown impressive advantages over SGE for the separation of DNA restriction fragments using either gel-filled capillaries 2 or polymeric solutions 3 and it is becoming widely accepted. Analysis and separation of DNA fragments has been performed by CE in a variety of ways.…”

Section: Introductionmentioning

confidence: 99%

“…They are the injection time and the applied voltage during the EK injection process. The quantity of the analytes injected (n a ) can be expressed by equation (3): 24,28 (3) where m a is the electrophoretic mobility of the analyte, µ eof is the EOF mobility, r is the inner radius of the capillary, Influence of the Electrokinetic Injection Conditions on the Separation of DNA Vol. 15, No.…”

Section: Introductionmentioning

confidence: 99%

Influence of the electrokinetic injection conditions on the separation of DNA fragments in capillary electrophoresis

Catai¹,

Carrilho²

2004

J. Braz. Chem. Soc.

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Em análises genéticas por eletroforese capilar com soluções poliméricas existem muitas variáveis que afetam a separação dos fragmentos de DNA. Uma das mais críticas é o processo da injeção da amostra, a qual pode afetar consideravelmente a eficiência e a resolução dos picos. Neste trabalho, estudou-se a influência da composição da amostra e as condições da injeção eletrocinética na separação dos fragmentos de DNA em eletroforese capilar com solução polimérica renovável. Os estudos foram realizados injetando-se eletrocineticamente as amostras do DNA sob uma variedade de condições tais como, a força iônica da amostra e sua capacidade tamponante e a qualidade da matriz de separação. Em todos os experimentos, os fragmentos de DNA foram corados com brometo de etídio para detecção por fluorescência induzida a laser e separados sob um campo elétrico de 200 V cm -1 em uma coluna capilar revestida com polialcoolvinílico e preenchida com uma solução 0,5% de hidroxietilcelulose como matriz de separação. Sob estas condições de análise, a composição da amostra deve conter entre 1 e 5 mmol L -1 de tampão de separação para a obtenção de injeções reprodutíveis com um coeficiente de variação menor que 4% tanto para o tempo de migração quanto para a quantidade de material injetado.In genetic analysis by capillary electrophoresis with polymer solutions there are many variables that affect separation of the DNA fragments. A very critical one is the sample injection process, which can considerably affect the peak efficiency and the resolution. In this work, we have studied the influence of the DNA sample composition and the electrokinetic injection conditions in the separation of DNA fragments by capillary electrophoresis using replaceable polymer solutions. The studies were carried out by electrokinetically injecting the DNA samples under a variety of conditions, such as sample ionic strength, buffering capacity and the quality of the separation matrix. In all experiments DNA was stained with ethidium bromide for laser-induced fluorescence (LIF) detection and separated in a poly(vinylalcohol) coated capillary column filled with 0.5% hydroxyethylcellulose solution under a 200 V cm -1 electric field. Under such conditions, samples prepared with 1 to 5 mmol L -1 of running buffer have been shown to produce reproducible injections with RSD < 4% for both migration time and peak area.

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Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

Cited by 399 publications

References 30 publications

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

DNA mutation detection via fluorescence imaging in a spatial thermal gradient, capillary electrophoresis system

Influence of the electrokinetic injection conditions on the separation of DNA fragments in capillary electrophoresis

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