Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis

Ueda, Mitsuru; Kiba, Yuriko; Abe, Hiromasa; Arai, Akihiro; Nakanishi, Hiroyuki; Baba, Yoshinobu

doi:10.1002/(sici)1522-2683(20000101)21:1<176::aid-elps176>3.0.co;2-a

Cited by 46 publications

(24 citation statements)

References 27 publications

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“…Similarly, Ueda et al [87] demonstrated fast separations (12 s analyses) of oligonucleotides and triplet repeat DNAs on microfabricated chips in separation channels of 6 mm length. A highly sensitive silicon-intensified target camera was adopted for imaging the fluorescently labelled DNA fragments.…”

Section: Microfluidic Chips For Dna Sizing and Quantitationmentioning

confidence: 99%

Recent progress in DNA analysis by capillary electrophoresis

Righetti¹,

Gelfi²,

D’Acunto³

2002

Electrophoresis

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A number of recent developments in DNA analysis by capillary electrophoresis are here reviewed. They include capillary arrays for fast, parallel DNA sequencing as well as microfabricated capillary arrays. Microfluidic chips for DNA sizing and quantitation are also covered, as well as microdevices containing arrays of regular obstacles acting as size-separators during DNA migration. Screening of DNA point mutations by two much improved techniques is also reported: in one case, such mutations are detected (but only on relative short, ca. 60-70 base-long fragments) by free electrophoresis in rather acidic (pH ca. 3) buffers; in the case of single-strand chain polymorphism, an improved technique is described based on near-neutral pH buffers with mixtures of Tris/MES cations/zwitterions. When studying the behavior of inorganic and organic cations in the Debye-Hückel layer of DNA, it was found that the latter (especially a large number of Good's buffers and other zwitterions, such as His) would bind to the DNA filament not only via charge interaction, but also via additional bonds, notably hydrogen bonds, thus altering the electrophoretic (and possibly the biological) behavior of DNA molecules. However, whether or not borate ions would bind to DNA remains still an unsettled question. Finally, capillary electrophoresis was found to be instrumental in measuring fine physicochemical parameters pertaining to DNA polyelectrolytes, such as their free mobility and their translational diffusion coefficients.

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Section: Microfluidic Chips For Dna Sizing and Quantitationmentioning

confidence: 99%

Recent progress in DNA analysis by capillary electrophoresis

Righetti¹,

Gelfi²,

D’Acunto³

2002

Electrophoresis

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“…The successful application of microchip electrophoresis techniques in DNA separations derives from their high speed, strong resolution, easy automation, and versatility for routine application [1][2][3][4][5][6]. The techniques also promise to be easily integrated with other components, e.g., polymerase chain reactions (PCRs) [7,8] and immobilized enzyme reactions [9], to solve complicated chemical and biological problems.…”

Section: Introductionmentioning

confidence: 99%

DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds

Jabasini

Baba

2002

Electrophoresis

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Low-viscosity polymer solutions have potential for double-stranded (ds) DNA separations in micrototal analysis systems (micro-TAS). In this paper, we report dilute, low-viscosity hydroxypropylmethylcellulose-50 (HPMC-50, 11.5 kDa) solutions containing polyhydroxy additives as separation media. Predominant operational variables, such as applied electric field strength, fluorescent intercalator (YOPro-1) concentration, polymer concentration, and additive concentration, are thoroughly investigated. Fast (within 170 s) and excellent separation of DNA restriction fragments ranging in size from 72 to 1353 base pairs (bp) is achieved in a 30 mm length channel of polymethylmethacrylate (PMMA) microchips at an electric field strength of 300 V/cm, by introducing 8% mannitol, 8% glucose or 10% glycerol additives into a 2% HPMC-50/1 x Tris-borate-EDTA (TBE) solution. The low-viscosity (40 cP) matrix formulation provides both coating of the microchannels and separation of DNA in one step. The performance in the solution surpasses that in highly concentrated HPMC-50 solution. In addition, separation using 1xTris-EDTA buffer in the 2% HPMC-50 matrix containing polyhydroxy additives also exhibits a notably increased performance. This is presumably due to formation of hydrogen-bonding interactions of polyhydroxy additives with HPMC-50 matrix and DNA so as to increase the coupling interactions between matrix and DNA molecules during electrophoresis. The result reflects that boric acid is not a prerequisite in polyhydroxy-enhanced HPMC-50 solution for separation.

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“…We have studied the electrophoretic behavior of triplet repeat DNA fragments by means of conventional capillary electrophoresis, and found that even a large CNG (N: C, A, T, G) repeat DNA moves much faster than a small random-sequence DNA in a polymer solution. [3][4][5] In this work, we performed the fast separation of a triplet repeat DNA fragment on a µ-CE (microfabricated-capillary electrophoresis) device. The µ-CE is a kind of device based on the idea of µ-TAS (micro total analytical system) or Lab-on-Chip (laboratory on a chip).…”

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confidence: 92%

Fast Separation of Oligonucleotide and Triplet Repeat DNA on a Microfabricated Capillary Electrophoresis Device

et al. 2000

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An extension for repeats of trinucleotide, like CTG, is known to cause some inherited neuromuscular diseases, called triplet repeat diseases. 1 Chastain et al. have reported that DNA fragments containing CTG repeats migrate up to 20% faster than expected in non-denaturing polyacrylamide gels. 2This unusual electric behavior of triplet repeat DNA is expected to be used for the detection of an extension of the triplet repeat. We have studied the electrophoretic behavior of triplet repeat DNA fragments by means of conventional capillary electrophoresis, and found that even a large CNG (N: C, A, T, G) repeat DNA moves much faster than a small random-sequence DNA in a polymer solution. [3][4][5] In this work, we performed the fast separation of a triplet repeat DNA fragment on a µ-CE (microfabricated-capillary electrophoresis) device. The µ-CE is a kind of device based on the idea of µ-TAS (micro total analytical system) or Lab-on-Chip (laboratory on a chip). 6 In the future, we plan to integrate on a chip all of the analytical methods for DNA diagnosis and gene therapy for human diseases, like the triplet repeat diseases. Firstly, we have investigated the separation of triplet repeat with chip-base, and demonstrated faster separation compared to conventional capillary electrophoresis. Experimental MaterialsWe used two kinds of synthetic single-stranded DNA fragments labeled by FITC with PAGE-purification grade. One was 30mer of a triplet repeat DNA fragment, 10 repeats of CTG. Another was 20mer of the sequence 5′-GTTGGCTCTGACTGTACCAC-3′, which was used as a DNA marker. These DNA fragments were dissolved with TB buffer (50 mM Tris-borate, pH 8.2). Methylcellulose (4000 cP at 2% solution) was used as a separation polymer. Figure 1 shows a schematic diagram of the µ-CE system. The microchip was made of two pieces of quartz plate (35 mm × 12 mm × 1 mm). The bottom plate had two orthogonal microchannels (50 µm wide and 20 µm deep), that ware generated by photolithography and chemical etching. The cover plate had four holes (1 mm in diameter) used as reservoirs (R1 -R4). The two plates were bonded by a treatment with a 1% hydrofluoric acid solution. 7 The inner wall of the channels was coated with linear polyacrylamide. The channels were filled † To whom correspondence should be addressed. E-mail: uedam@fc.ph.tokushima-u.ac.jp Fig. 1 Schematic diagram of the µ-CE system with a laser-induced fluorescence detector. Apparatus

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Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis

Cited by 46 publications

References 27 publications

Recent progress in DNA analysis by capillary electrophoresis

Recent progress in DNA analysis by capillary electrophoresis

DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds

Fast Separation of Oligonucleotide and Triplet Repeat DNA on a Microfabricated Capillary Electrophoresis Device

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