Article

Sequence-selective DNA detection using multiple laminar streams: A novel microfluidic analysis method

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Abstract

On-site detection methods for DNA have been demanded in the pathophysiology field. Such analysis requires a simple and accurate method, rather than high-throughput. This report describes a novel microfluidic analysis method and its application for simple sequence-selective DNA detection. The method uses a microchannel device with a serpentine structure. Sequence-specific binding of probe DNA can be detected at one side of the microchannel. This method is capable of sequence-specific detection of DNA with high accuracy. Single base mutations can also be analyzed. Combination of laminar stream and laminar secondary flow in the microchannel enable specific detection of probe-bound DNA.

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... Such a process could be performed in solution or probe could be presented on a solid support. Their detection could be electrochemical [2,3] or optical [4][5][6][7][8]. However, in majority of the cases, nucleic acid is covalently labeled by a fluorescent probe and the detection is studied by fluorescence resonance energy transfer (FRET) or fluorescence quenching. ...
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Fluorescence resonance energy transfer has been used to illustrate its applicability to the sensitive detection of DNA hybridization reactions in a PDMS microfluidic channel.
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The expression of many genes involved in xenobiotic/drug metabolism and transport is regulated by at least three nuclear receptors or xenosensors: aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR). These receptors establish crosstalk with other nuclear receptors or transcription factors controlling signaling pathways that regulate the homeostasis of bile acids, lipids, glucose, inflammation, vitamins, hormones, and others. These crosstalks are expected to modify profoundly our vision of xenobiotic/drug disposition and toxicity. They provide molecular mechanisms to explain how physiopathological stimuli affect xenobiotic/drug disposition, and how xenobiotics/drugs may affect physiological functions and generate toxic responses. In addition, the possibility that xenosensors may control other signaling pathways opens the way to new pharmacological opportunities.
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FXR (farnesoid X receptor), a nuclear receptor activated by BAs (bile acids), is a key factor in the regulation of BA, lipid and carbohydrate metabolism. The recent development of synthetic FXR agonists and knockout mouse models has accelerated the discovery of FXR target genes. In the present study, we identify human fetuin-B as a novel FXR target gene. Treatment with FXR agonists increased fetuin-B expression in human primary hepatocytes and in the human hepatoma HepG2 cell line. In contrast, fetuin-B expression was not responsive to FXR agonist treatment in murine primary hepatocytes. Fetuin-B induction by FXR agonist was abolished upon FXR knockdown by siRNA (small interfering RNA). In addition to the previously described P1 promoter, we show that the human fetuin-B gene is also transcribed from an alternative promoter, termed P2. Transcription via the P2 promoter was induced by FXR agonist treatment, whereas P1 promoter activity was not sensitive to FXR agonist treatment. Two putative FXR-response elements [IR-1 (inverted repeat-1)] were identified in the region -1.6 kb upstream of the predicted P2 transcriptional start site. Both motifs bound FXR-RXR (retinoid X receptor) complexes in vitro and were activated by FXR in transient transfection reporter assays. Mutations in the IR-1 sites abolished FXR-RXR binding and activation. Taken together, these results identify human fetuin-B as a new FXR target gene in human hepatocytes.
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The application of micro total analysis system (microTAS) has grown exponentially in the past decade. DNA analysis is one of the primary applications of microTAS technology. This review mainly focuses on the recent development of the polymeric microfluidic devices for DNA analysis. After a brief introduction of material characteristics of polymers, the various microfabrication methods are presented. The most recent developments and trends in the area of DNA analysis are then explored. We focus on the rapidly developing fields of cell sorting, cell lysis, DNA extraction and purification, polymerase chain reaction (PCR), DNA separation and detection. Lastly, commercially available polymer-based microdevices are included.
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We investigate the analytical performance of an interdigitated electrode sensor for the label-free detection of DNA, by monitoring the complex impedance of 5 microm wide interdigitated Pt microelectrodes on a glass substrate. We detect the hybridization of unlabeled 38-mer target ssDNA with a complementary probe that is bound on the glass in between the electrodes by a disuccinimidyl terephtalate and aminosilane immobilization procedure. The sensor is mounted in a microfluidic flow cell, in which hybridization is monitored and in situ compared with a reference. After hybridization, the cell is perfused with deionised water and the dependence of the measured conductance due to the immobilized target DNA layer, to target DNA concentrations down to 1 nM is demonstrated. Subsequently, we apply our sensor to the detection of pathogen DNA from Salmonella choleraesuis in dairy food.
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A method for inline measurement of composition of binary liquid mixture in a nanoliter volume is presented. Low-coherence interferometric technique, based on fiber-optic Mach-Zehnder interferometer, is applied for measuring the liquid refraction index, from which its volume fractions are found. The accuracy of volume fractions measurement, of about , was predominantly determined by the accuracy of reading the position of mechanical scanner. The data rate of about 1.5 Hz was also limited by mechanical scanning.
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Most fluids show laminar behavior in miniature flow structures with channel cross-sections below 0.5 mm. Different layers of miscible fluids and particles can flow next to each other in a microchannel without any mixing other than by diffusion. Small particles diffuse faster than larger ones, which allows separation of particles by size. It is possible to design fluidic microchips in which separations, chemical reactions, and calibration-free analytical measurements can be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples.
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In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition.
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Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 × 0.1 × 0.02 mm or 1 × 1 × 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of ‘microtest tube’ of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.
Article
A synthesized 20-mer DNA probe complementary to a part of an oncogene v-myc region having a mercaptohexyl group at the 5'-phosphate end was immobilized on a gold electrode by chemisorption. The immobilized DNA was detected voltammetrically using Hoechst 33258 with a DNA minor groove binder and an electrochemically active dye. The modified electrode was immersed into a 100 mumol/L Hoechst 33258 solution and washed with a phosphate buffer (pH 7.0). The anodic peak current (ipa) of Hoechst 33258 on the modified electrode was higher than that on a bare gold electrode (128 and 75 nA, respectively). It was considered that Hoechst 33258 was concentrated on the electrode surface due to its association with DNA. When the modified electrode was hybridized in a solution of a model targeted gene (10(-7) g/mL), single-stranded pVM623 containing the PstI fragment of a 1.5-kilobase pair of oncogene v-myc, the ipa was 192 nA. On the other hand, the ipa was 128 nA when the modified electrode was reacted in a solution of single-stranded pUC119 without a region complementary to v-myc in pVM623. The ipa was related to the concentration of the targeted DNA in the hybridization reaction. The use of Hoechst 33258 resulted in a sequence-specific detection of the targeted DNA quantitatively ranging from 10(-7) to 10(-13) g/mL in a buffer solution.
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Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA. On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode. Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s. The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode. Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level. The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol.
Article
Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to [Fe(CN)6]3-. The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without stringent hybridization conditions. Furthermore, many common DNA lesions and "hot spot" mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.
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  • W G Crook
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  • Nature
  • Genet
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