Mr. Steve
R. Karsjen
111 TASF
Ames Laboratory
Iowa State University
Ames, IA 50011
Phone: 515-294-1856
Fax: 515-294-3226
E-mail: karsjen@ameslab.gov
Number of Human Subjects projects reported: 2
"Single Molecule DNA Assay and Immunoassay"
Principal Investigator: Dr. Edward S. Yeung, Ames Laboratory
Project started in: 1998
Funding for Human Subjects Research:
This project does not involve the use of multiple protocols/subprojects.
Institutional Review Board (IRB) Review:
Type of Review: Full Board
Approving Institution: Iowa State University
Most recent approval: 06/15/00
Number of human subjects who participated in this project/protocol/subproject in the last reporting period: 9
Reporting period for number of human subjects:
Fiscal Year 2000 (10/1/99-9/30/2000)
Type(s) of Human Subjects Involvement:
We will develop novel imaging technologies for real-time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g. DNA region, RNA region, protein), and to do so at relatively low cost. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. The general scheme is based on novel concepts for single-cell and single-molecule detection and characterization recently demonstrated in our laboratory. We are therefore in a unique position to respond to this particular RFA. Four distinct but interrelated goals are identified: (1) development of a diffusion-based single-molecule detection system which permits rapid and highly confident detection of hybridization to a DNA probe or binding to an antibody in the presence of a large excess of unhybridized probe molecules or free anti-bodies, respectively. This will lead to single-event homogeneous assays of molecular alterations of DNA and proteins, respectively, in biological tissues; (2) development of a microscale single-molecule electrophoresis system for the rapid detection and identification of specific targets in the presence of a large excess of similar species. This will extend single-molecule homogeneous assays to small molecules relevant to molecular profiling. It may even be possible to detect directly targeted species in biological tissues without introducing additional probes; (3) development of high-speed high-throughput cell screening protocols based on the above concepts and specialized data treatment software for rapid "on-line" analysis of the images with an emphasis on confidence in target recognition and reduction in post-imaging data work-up; (4) demonstration of actual analysis of human cells and tissue samples, with out-side collaboration, to evaluate and to validate the technology developed, and to further optimize the performance regarding speed, ruggedness and accuracy. Initially, we plan to target one model virus and one known region of a DNA.
Only human tissues (blood, cheek cells, and commercial human cell lines) will be used. No individuals will be identified with the samples.
"High-Speed High-Throughput Mutation Detection"
Principal Investigator: Dr. Edward S. Yeung, Ames Laboratory
Project started in: 1999
Funding for Human Subjects Research:
This project does not involve the use of multiple protocols/subprojects.
Institutional Review Board (IRB) Review:
Type of Review: Full Board
Approving Institution: Iowa State University
Most recent approval: 06/15/00
Number of human subjects who participated in this project/protocol/subproject in the last reporting period: 9
Reporting period for number of human subjects:
Fiscal Year 2000 (10/1/99-9/30/2000)
Type(s) of Human Subjects Involvement:
Single-nucleotide polymorphism (SNP) detection has been the focus of much attention recently. Although many methods have been reported, low-cost, high-throughput and high-detection-rate methods are still in demand. We present a fast and reliable mutation detection scheme based on temperature-gradient capillary electrophoresis. A large temperature gradient (10 °C) was applied with a precision of 0.02 °C and a temperature ramp of 0.7 °C/min. Multiple unlabeled samples from PCR reaction were injected and analyzed. Ethidium bromide was used as the intercalating dye for laser-induced fluorescence detection. Mutations can be recognized by comparing the electrophoretic patterns of the heteroduplex with that of a homoduplex reference without prior knowledge of the exact type of mutation present. This scheme is demonstrated in 96-capillary array electrophoresis for screening single-point polymorphism in large numbers of samples prior to full sequencing of only the positive samples to identify the nature of the mutation.
Only human tissues (blood, cheek cells, and commercial human cell lines) will be used. No individuals will be identified with the samples.