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Rapid Separation of Megabase Sized DNA in Nanostructures Open Access


Other title
nanoporous sieving matrix
megabase DNA separation
colloidal self-assembly
asymmetric pulsed field
intermittent field
Type of item
Degree grantor
University of Alberta
Author or creator
Sheng, Huiying
Supervisor and department
Harrison, D. Jed (Chemistry)
Examining committee member and department
Freeman, Mark (Physics)
Gibbs-Davis, Juli (Chemistry)
Buriak, Jillian (Chemistry)
Campbell, Robert (Chemistry)
Soper, Steven (Chemistry)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
This thesis reports the use of high magnitude asymmetric pulsed fields and high frequency zero-field interruptions on the pulses to accomplish angular separation of megabase and submegabase sized DNA in colloidal self-assembled silica particle arrays. First, a simple particle array packing strategy is developed to assemble large silica particles in microfluidic devices without being interrupted by particle sedimentation. The strategy also selectively shields the injection channel from particle packing to allow fast and efficient injection of megabase DNA into the particle-packed separation bed for analysis such as DNA trapping and DNA separation. Then, trapping studies of bulk sample and single DNA molecules have been conducted under the asymmetric pulsed fields in particle arrays, where a notably high field magnitude allowed for separation has been identified for DNA sizes up to 0.9 Mbp. With the zero-field interruptions, further reduction in trapping has been observed. Following this finding, separation of DNA sizes up to 0.9 Mbp has been performed with the intermittent asymmetric pulses at over 100 V/cm, speeding up the separation enormously relative to current pulsed field gel electrophoresis for megabase DNA separation. Separation optimization indicates a better performance in smaller pores for particle arrays; in contrast with the trend for pulse field gel electrophoresis favoring more dilute gels. With a nonmonotonic relation between deflection angle and pulse frequency for DNA, average molecular lengths have been deduced from the deflection-angle-frequency relation, leading to a pore size dependence departing from the de Gennes theory that accounts for DNA confined in such sized pores. This conflict of DNA lengths in different sized pores has also been observed by a single molecule imaging study, which matches the average lengths deduced from bulk flow separation results, indicating different features between separation in particle arrays and in gels. Finally, a geometry model with fitting parameters accounting for useful physical meanings has been developed to describe the relation between deflection angle and frequency obtained from bulk flow separation, delivering a new means to understand and improve separation performance.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
Huiying Sheng et al. 2013

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