Abstract

Using fast video microscopy and electrophoresis we will investigate of the mechanism of pulsed field electrophoresis of DNA in polymer solutions. We will test the hypothesis that pulsed field electrophoresis operates to reduce the range of entanglement times of DNA and the entangling polymer. Proposed synchronization mechanisms will be tested by cross-correlation of individual molecule velocities and extensions, and by fluorescence anisotropy imaging of orientable labeled dextrans. Similar studies will be performed to elucidate the electrophoretic dynamics of sc-plasmid DNA and topoisomerases will be used to generate individual topoisomers for detailed study of band broadening. Three-dimensional video microscopy based on fast serial sectioning will be used to elucidate the migration dynamics in detail. Size exclusion electrochromatographic separation techniques will be developed for lightly charged or uncharged plasmid complexes. Prior electrophoretic separation may be used for plasmids and ds-DNA. Applications and mechanisms of electrophoresis in ultraconcentrated polyacrylamide solution will be investigated. Pulsed field electrophoresis will be used to improve the resolution in the 100 kbp - 1 Mbp range and video microscopy to investigate the unusual dynamics in ultraconcentrated polyacrylamide and related media. Additionally, analyte velocity modulation will be explored as a fluorescence background rejection technique for protein separations in plastic microchips. The electrophoresis driving voltage will be modulated at low frequency to generate a modulation component on analyte fluorescence. The concept will be tested on model protein separations, such as the serum proteins or commercial size standards.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037006-14
Application #
6179757
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Edmonds, Charles G
Project Start
1986-07-01
Project End
2003-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
14
Fiscal Year
2000
Total Cost
$148,537
Indirect Cost

  Institution

Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109

  Publications

Rhee, Minsoung; Burns, Mark A (2009) Microfluidic pneumatic logic circuits and digital pneumatic microprocessors for integrated microfluidic systems. Lab Chip 9:3131-43
Rhee, Minsoung; Burns, Mark A (2008) Microfluidic assembly blocks. Lab Chip 8:1365-73
Rhee, Minsoung; Burns, Mark A (2008) Drop mixing in a microchannel for lab-on-a-chip platforms. Langmuir 24:590-601
Al-Hashimi, Hashim M; Walter, Nils G (2008) RNA dynamics: it is about time. Curr Opin Struct Biol 18:321-9
Walter, Nils G; Huang, Cheng-Yen; Manzo, Anthony J et al. (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475-89
Walter, Nils G (2007) Ribozyme catalysis revisited: is water involved? Mol Cell 28:923-9
Yamaguchi, Yoshinori; Todorov, Todor I; Morris, Michael D et al. (2004) Distribution of single DNA molecule electrophoretic mobilities in semidilute and dilute hydroxyethylcellulose solutions. Electrophoresis 25:999-1006
Todorov, Todor I; Yamaguchi, Yoshinori; Morris, Michael D (2003) Effect of urea on the polymer buffer solutions used for the electrophoretic separations of nucleic acids. Anal Chem 75:1837-43
Todorov, Todor I; Morris, Michael D (2002) Comparison of RNA, single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions. Electrophoresis 23:1033-44
de Carmejane, O; Yamaguchi, Y; Todorov, T I et al. (2001) Three-dimensional observation of electrophoretic migration of dsDNA in semidilute hydroxyethylcellulose solution. Electrophoresis 22:2433-41

Showing the most recent 10 out of 43 publications