A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of `third generation' instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.
A glass microchip column was fabricated for free-solution electrophoresis. The channels were wet chemically etched on a substrate using standard photolithographic techniques and were sealed using a direct bonding technique. Two methods * Chemical and Analytical Sciences Division.
Fast, efficient separations are sought in liquid-phase analyses which incorporate a nondiscriminatory sample injection scheme and can implement a variety of detection modes. A glass microchip device for free solution electrophoresis was fabricated using standard photolithographic procedures and chemical wet etching. Separations were performed at several separation lengths from the injector to the detector with electric field strengths from 0.06 to 1.5 kV/cm. For a separation length of 0.9 mm, electrophoretic separations with baseline resolution are achieved in less than 150 ms with an electric field strength of 1.5 kV/cm and an efficiency of 1820 plates/s. For a separation length of 11.1 mm, a minimum plate height of 0.7 jtm and a maximum number of plates per second of 18 600 were achieved.Microfabricated chemical instruments show great promise for the laboratory and as advanced chemical sensors. Microfabrications of chemical separation techniques have received noticeable attention over the past several years and have included the techniques of gas chromatography,1 liquid chromatography,1 2 3456and capillary electrophoresis.3-7 Microelectronic devices have been able to achieve even faster response times in part due to miniaturization. Similar benefits may also accrue from miniaturization of some chemical measurement techniques. The response times of chemical measurements can often be an important issue, in particular in the case of chemical sensing.With microfabrication, the performance of many liquid separation techniques improves, especially capillary electrophoresis.8-10 For capillary electrophoresis, smaller column dimensions enable the power generated to be dissipated more efficiently, and as a direct result, separation devices can be operated at higher electric field strengths. The efficiency of the separations, therefore, improves in two areas. First, Joule heating, which leads to thermal gradients within the channel and ultimately contributes to the dispersion of the analyte
An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.
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