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Micellar and chromatographic media f...

Savard, Jeffrey Michael.

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Micellar and chromatographic media for surfactant-based DNA analysis and purification processes.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Micellar and chromatographic media for surfactant-based DNA analysis and purification processes./
Author:	Savard, Jeffrey Michael.
Description:	363 p.
Notes:	Source: Dissertation Abstracts International, Volume: 69-03, Section: B, page: 1790.
Contained By:	Dissertation Abstracts International69-03B.
Subject:	Chemistry, Analytical. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3305805
ISBN:	9780549525196

Micellar and chromatographic media for surfactant-based DNA analysis and purification processes.
Savard, Jeffrey Michael.

Micellar and chromatographic media for surfactant-based DNA analysis and purification processes. - 363 p.

Source: Dissertation Abstracts International, Volume: 69-03, Section: B, page: 1790.

Thesis (Ph.D.)--Carnegie Mellon University, 2008.

We develop and extend the dynamic range of surfactant-based strategies for sequence and length-dependent nucleic acid purification. DNA is alkylated in solution and then is separated by leveraging hydrophobicity differences between labeled and unlabeled strands. Solution-based modification alleviates most limitations of surface-based techniques.

ISBN: 9780549525196Subjects--Topical Terms:

586156
Chemistry, Analytical.

Micellar and chromatographic media for surfactant-based DNA analysis and purification processes.
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100 1 $a Savard, Jeffrey Michael. $3 1278973
245 1 0 $a Micellar and chromatographic media for surfactant-based DNA analysis and purification processes.
300 $a 363 p.
500 $a Source: Dissertation Abstracts International, Volume: 69-03, Section: B, page: 1790.
502 $a Thesis (Ph.D.)--Carnegie Mellon University, 2008.
520 $a We develop and extend the dynamic range of surfactant-based strategies for sequence and length-dependent nucleic acid purification. DNA is alkylated in solution and then is separated by leveraging hydrophobicity differences between labeled and unlabeled strands. Solution-based modification alleviates most limitations of surface-based techniques.
520 $a DNA is modified by one of two routes, depending on the application: direct alkylation by covalent tagging or alkylation by peptide nucleic amphiphile (PNAA) hybridization. Covalent alkylation allows for the direct creation of labeled single-stranded and double-stranded DNA of high purity through polymerase chain reaction, and is used for techniques in which sequence-specificity is not needed, such as DNA sequencing. A covalent tag also allows us to operate under denaturing conditions to create alkylated, single-stranded DNA. In contrast, PNAA hybridization permits the sequence-specific labeling of target strands from complex mixtures. This alkylation method is useful for isolation of specific strands from non-specific nucleic acid pools, such as in quantification of gene expression. We utilize PNA for sequence recognition due to its superior DNA hybridization properties relative to DNA.
520 $a We analyze hydrophobic DNA created via both alkylation methods in micellar electrokinetic chromatography (MEKC) with Triton X-100 micelles. We demonstrate that native DNA does not interact with the micellar sub-phase, while alkylation of DNA leads to powerful and tunable micelle partitioning. The differential partitioning of labeled and unlabeled strands leads to their rapid separation in MEKC. We model the electrophoretic properties of a hydrophobic DNA with an equilibrium binding model that accurately describes the effect of Triton X-100 micelle concentration on DNA separations in MEKC. We observe that the use of our most hydrophobic modifications results in near-complete partitioning of the DNA into the micellar sub-phase. Using covalent DNA alkylation with a single tag, we demonstrate facile separation of DNA up to 500 bases or base pairs from unlabeled fragments. Dual-micelle tagging of double-stranded DNA dramatically extends this separation window to over 700 bp. Using alkylation by hybridization and dual micelle-tagging, we achieve separation of a 1000 base ssDNA from unlabeled fragments, which to our knowledge is the longest free-solution electrophoretic separation of DNA published. We utilize the theoretical framework of end-labeled free solution electrophoresis (ELFSE) to quantify the effect of transient micelle tagging on DNA electrophoretic velocity. The effective drag added to a DNA strand from micelle attachment is equal to or superior to any covalent tagging scheme previously reported, highlighting the potential of this technique in length-based DNA separations.
520 $a We demonstrate tunable and predictable control over DNA-micelle partitioning through tail length variation. Using four different alkane lengths in conjunction with a low micelle concentration, we achieve multicomponent separations via differential partitioning. We invoke a free energy argument to describe the partitioning process, and calculate the transfer energy of a methylene group into a micelle, discovering that it is in close agreement with published results describing similar processes. Partitioning is also found to be independent of electric field strength. These findings indicate that DNA partitioning to a micellar sub-phase is an equilibrium process.
520 $a We investigate the use of alternative surfactant microstructures for DNA separations using MEKC. We demonstrate that DNA analysis in MEKC is compatible with several distinct surfactant classes, including a non-ionic fluorosurfactant, which had not been previously reported. We find that micelle size, shape, and charge all have profound impacts on the separation efficacy. We identify a new surfactant, C16E6, which forms giant worm-like micelles in solution and in certain cases outperforms Triton X-100.
520 $a We utilize a group-based chromatography technique, hydrophobic interaction chromatography, for the sequence-specific separation of target DNA from complex mixtures. We demonstrate that PNAA is capable of binding target DNA in solution, and the duplex can be readily separated by its heightened retention on HIC media relative to native strands. We achieve baseline resolution between a 60 base single-stranded DNA and two non-target strands each featuring a single mismatch. The HIC method is extended to purification of double-stranded DNA by using a PNAA capable of strand invasion. We demonstrate that alkylation does not hinder dsDNA binding relative to PNA itself. We also develop a batch HIC technique that permits the parallel, high-throughput processing of alkylated ssDNA and dsDNA samples.
590 $a School code: 0041.
650 4 $a Chemistry, Analytical. $3 586156
650 4 $a Engineering, Chemical. $3 1018531
690 $a 0486
690 $a 0542
710 2 $a Carnegie Mellon University. $3 1018096
773 0 $t Dissertation Abstracts International $g 69-03B.
790 $a 0041
791 $a Ph.D.
792 $a 2008
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3305805