The proposed research involves the characterization and development of sinusoidal voltammetry for genomic mapping, sequencing and analysis of DNA. Sinusoidal voltammetry at copper microelectrodes (electrodes of micron dimensions) will provide a new generation of technology that will enable a dramatic reduction in the cost and time in traditional approaches to DNA sequencing, as well as provide a new format for resequencing and genomic mapping, once it is integrated into a chip format. Three primary aspects of this analysis will be addressed - 1) fundamental processes which can be used at all levels to enhance performance, 2) the implementation of SV for the analysis of nucleotides in conduction with capillary separations and traditional format dideoxy sequencing technologies, and most importantly, 3) the utilization of SV for the analysis of DNA after hybridization with specific probe sequences, enabling on-chip sequencing by hybridization and detection. Ultimately, it is hoped to miniaturize these concepts to the micron-scale to produce the next generation of chip-level devices capable of detecting down to a few hundred molecules of a specific DNA sequence using a total sample volume of less than 10 microliters. Sinusoidal voltammetry monitors the voltammetric current from complex electrochemical waveforms in the frequency domain. This information can be used to discriminate the Faradaic response from background currents and between redox species with different electrochemical properties (e.g., E', Eswitch, scan rate, number of electrons, electron transfer rate constant, etc.). Selectivity can be enhanced through selection of the appropriate frequency and phase to monitor a specific electroactive species. These methods lead to ultrasensitive and selective detection of DNA and nucleic acids. The protocol allows the detection of DNA with nanogram/mL sensitivity (corresponding to attomole/mL), which is comparable to or exceeds the sensitivity of conventional techniques which employ radioactivity or fluorescence measurements. This sensitivity is obtained without extensive sample pretreatment and employs instrumentation that is relatively inexpensive, small, and simple to operate. This may make it amenable for use in the biotechnology industry for quantification of DNA samples (including DNA sequencing after size-based separations or after hybridization with appropriate DNA probes, etc.), clinical applications (after miniaturization to produce credit-card sized sensor arrays for the detection of pathogenic bacteria, genetic diseases and forensics) and general application in pharmaceutical testing and quality control. The process is also easily adapted for addition to commercially available chromatographic and electrophoretic instruments, allowing the detection of these species after a chemical separation step.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HG001828-01
Application #
2707233
Study Section
Special Emphasis Panel (ZHG1-HGR-P (M1))
Project Start
1998-07-01
Project End
2001-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California Riverside
Department
Chemistry
Type
Schools of Earth Sciences/Natur
DUNS #
City
Riverside
State
CA
Country
United States
Zip Code
92521
Hebert, Nicole E; Kuhr, Werner G; Brazill, Sara A (2003) A microchip electrophoresis device with integrated electrochemical detection: a direct comparison of constant potential amperometry and sinusoidal voltammetry. Anal Chem 75:3301-7
Zhao, Dong S; Roy, Binayak; McCormick, Matthew T et al. (2003) Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining. Lab Chip 3:93-9
Brazill, Sara; Hebert, Nicole E; Kuhr, Werner G (2003) Use of an electrochemically labeled nucleotide terminator for known point mutation analysis. Electrophoresis 24:2749-57
Hebert, Nicole E; Kuhr, Werner G; Brazill, Sara A (2002) Microchip capillary electrophoresis coupled to sinusoidal voltammetry for the detection of native carbohydrates. Electrophoresis 23:3750-9
Brazill, Sara A; Kuhr, Werner G (2002) A single base extension technique for the analysis of known mutations utilizing capillary gel electrophoreisis with electrochemical detection. Anal Chem 74:3421-8
Brazill, S A; Kim, P H; Kuhr, W G (2001) Capillary gel electrophoresis with sinusoidal voltammetric detection: a strategy to allow four-""color"" DNA sequencing. Anal Chem 73:4882-90
Brazill, S A; Singhal, P; Kuhr, W G (2000) Detection of native amino acids and peptides utilizing sinusoidal voltammetry. Anal Chem 72:5542-8
Liu, Y; Kuhr, W G (1999) Separation of double- and single-stranded DNA restriction fragments: capillary electrophoresis with polymer solutions under alkaline conditions. Anal Chem 71:1668-73