The project will investigate fundamental phenomena in electrophoresis gels and apply these results to novel forms of gel electrophoresis and detection of nucleic acids and proteins in gels. Both slab gel and capillary formats will be investigated. The dynamics of gel compression and extension will be investigated using electric birefringence and fluorescence polarization imaging of slab gels and gel-filled capillaries. The effects of dynamic gel distortion on electrophoretic mobilities of nucleic acids will be investigated, and used to propose an improved separation model. Electric birefringence will be used for stainless and non-nicking imaging of agarose and polyacrylamide electrophoresis gels. Numerical techniques will be used to remove artifacts and background from the images to achieve subnanogram sensitivity. On-line imaging will be used for real-time observation and to control pulse parameters in several forms of pulse-field electrophoresis, with the goal of optimizing separations and minimizing running times. Electric birefringence will also be used as a detector principle for nucleic acids in pulsed field capillary gel electrophoresis. Diode laser excitation and lock-in amplifier extraction of birefringence will be used to achieve high detection sensitivity. Pulsed field capillary gel electrophoresis will be developed. In conventional quartz capillaries, the effect of different pulsing regimes will be studied. Agarose-loaded cellulose acetate capillaries will be used with external electrodes to implement orthogonal field capillary gel electrophoresis and related techniques, which are expected to extend the utility of CGE to the 100 kbp size range or higher. Raman microspectrometry will be used to investigate structural changes in polyacrylamide and other gels in capillaries, to determine the factors governing capillary life times.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037006-09
Application #
2178627
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1986-07-01
Project End
1995-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
9
Fiscal Year
1994
Total Cost
Indirect Cost
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
Rhee, Minsoung; Burns, Mark A (2009) Microfluidic pneumatic logic circuits and digital pneumatic microprocessors for integrated microfluidic systems. Lab Chip 9:3131-43
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
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
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

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