Temporal analysis of DNA restriction digests by capillary electrophoresis

Stebbins, Michael; Schar, Christine R.; Peterson, Cynthia B.

doi:10.1016/s0378-4347(97)00227-2

Cited by 12 publications

(10 citation statements)

References 14 publications

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“…A 1-cm long window was prepared at 8 cm from the detection end of the capillary by removing the outer polyamide coating by flame. Subsequently, the capillary walls were coated with linear polyacrylamide to eliminate electroosmotic flow and surface adsorption (8). Columns prepared in this manner were usually used for numerous runs over a 2-to 4-week period.…”

Section: Methodsmentioning

confidence: 99%

“…The CE-LIF detection apparatus was assembled in-house as described in detail previously (8). Briefly, a helium-neon greenie laser (1.5 mW, Edmund Scientific, Barrington, NJ) was used to generate an excitation beam at 543.5 nm.…”

Section: Methodsmentioning

confidence: 99%

“…The technique has experienced widespread use in biochemistry and molecular biology. For example, it has been used for DNA sequencing (2)(3)(4), studies of protein-nucleic acid interactions (5), analysis of the products of the polymerase chain reactions (6,7), and analysis of DNA restriction digests (8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

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Capillary Electrophoresis as a Method to Study DNA Reassociation

White

Stokes

et al. 2001

Biotechnology Progress

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To develop analytical methodology to assess the genetic complexity of a DNA sample, capillary electrophoresis with laser-induced fluorescence detection is used to monitor the annealing process of DNA samples. Coated columns are filled with an entangled polymer solution shown to optimally separate DNA through size-selective capillary electrophoresis. DNA samples are denatured by heating in a boiling water bath for approximately 10 min and then cooled to approximately 25 degrees C below the melting point of the DNA sample to initiate the reassociation process. The DNA is detected by means of the laser-induced fluorescence of intercalated ethidium bromide, which produces a substantially greater signal for double- versus single-stranded DNA. The rate of reassociation is dependent upon the rate at which complimentary strands of DNA encounter each other and the degree of repeating base sequences in the sample (hence, the diversity of the DNA). Experimental parameters also influence the reassociation rate. The effects of salt concentration and incubation temperature are presented. Traditional plots of C(o)t (C(o) = DNA concentration and t = reassociation time) versus % recovery of double-stranded DNA signal are generated for PhiX 174 Hae III digest and 50 bp stepladder DNA, individually and combined, to calculate the reassociation rate constants for these samples. Because reassociation of individual fragments is observed by the CE-LIF method, more information about the samples is available than with less specific and time-consuming traditional methods of investigating DNA reassociation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Capillary Electrophoresis as a Method to Study DNA Reassociation

White

Stokes

et al. 2001

Biotechnology Progress

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“…CE has been used in research which relates to nuclease and nucleic acids. [23][24][25][26][27][28] Comparing with CE, MCE, which is a modern trace analysis technique, has attracted increasing attention owing to its numerous advantages such as lower sample and reagent consumption, high separation efficiency, portability and the possibility of being fully automated. [29][30][31][32] However, since the channels in microuidic devices are very narrow, a highly sensitive detection mode is required for biochemical and clinical analysis.…”

Section: Introductionmentioning

confidence: 99%

Ultrasensitive nuclease activity and inhibition assay using microchip electrophoresis with laser induced fluorescence detection

et al. 2016

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“…Compared with traditional electrophoresis method, capillary electrophoresis (CE) is a modern separation and analytical technique that has rapidly developed in recent years. CE has many advantages (such as high separation efficiency, high speech, small sample volume requirements, and low analytical cost) especially when combined with laser-induced fluorescence detector (LIFD), which makes its highly sensitive [1]. Based on the polymorphism of DNA segments after enzyme digestion, CE can be used in the analysis of the homeosis of genome and class identifications of individuals.…”

Section: Introductionmentioning

confidence: 99%

Restriction Enzyme Pattern Analysis of Mycobacteria DNA by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Wang

et al. 2006

Front. Chem. China

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A new method for rapidly detecting restriction enzyme patterns of Mycobacterium DNA using capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) was developed. Polymerase chain reaction was used to amplify a 439-bp fragment of a 65,000-kDa (M r ) heat shock protein gene (hsp65) of Mycobacterium. After digesting amplification products by BstEII and HaeIII, patterns of enzyme cleavage products were detected by both CE-LIFD and agarose gel electrophoresis (AGE), respectively. Experimental parameters of CE were optimized. Restriction enzyme patterns of Mycobacterium DNA were detected in optimum electrophoresis conditions: a coated capillary column with a length of 50 cm and an internal diameter of 100 μm, an electrophoresis buffer of 45 mmol/l Tris-boric acid-ethylenediaminetetraacetic acid, and a running voltage of 11 kV. The restriction enzyme patterns for eight species of mycobacteria were studied. Relative standard deviations of the relative migration times of DNA segments were <3.6%. Compared with AGE, CE is more outstanding in resolution and detection time, and it can be applied as a more effective means to DNA restriction enzyme pattern analysis.

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Temporal analysis of DNA restriction digests by capillary electrophoresis

Cited by 12 publications

References 14 publications

Capillary Electrophoresis as a Method to Study DNA Reassociation

Capillary Electrophoresis as a Method to Study DNA Reassociation

Ultrasensitive nuclease activity and inhibition assay using microchip electrophoresis with laser induced fluorescence detection

Restriction Enzyme Pattern Analysis of Mycobacteria DNA by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

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