Gel-Free Separation and Detection of DNA via Micelle-tagging Electrophoresis in Entangled Micelle Networks

Our group has developed a novel, gel-free DNA separation technique called Micelle-Tagging Electrophoresis (MTE). MTE achieves rapid, high resolution DNA separation in free solution by transiently attaching non-ionic micelles to DNA as drag-tags via end-alkylation. This thesis details our efforts in optimizing, improving and extending MTE to various applications of DNA separation. We demonstrate the first kilobase Sanger sequencing separation using MTE by symmetrically induce micellar growth, achieving a read length of 1150 bases with a buffer composed of 150mM C12E5 and 3M urea at 33°C. This is more than four times improvement than other gel-free separation techniques using covalently attached drag-tags. However, the lack of larger drag tags and a reliable method for end-alkylation of long DNA prevented the study of kilobase DNA separation via MTE. Addressing these challenges, we develop a reliable and facile method for end-alkylation of kilobase DNA using sequence specific alkylated γPNA probes. We also significantly increase the size of these micelles using ternary CiEj surfactant systems consisting of C16E6, C12E5 and C10E5. However, as these micelles grow longer, they become entangled and form transient networks that mimic polymer gels with an opposite elution order as MTE, leading to different deviations (ssDNA vs dsDNA) from the standard unsegregated MTE theory. We investigate the cause of these deviations in kilobase ssDNA and determine it to be due to hydrodynamic segregation of DNA-micelle complex. More specifically, the degree of segregation is dependent on the strength of the applied electric field and not due to background sieving of entangled micelle network or DNA’s steric limitation. We also show that entangled micelles can be used as a separation medium for untagged dsDNA. For the first time, both untagged and tagged kilobase dsDNA up to 23kB were separated simultaneously in under 5 minutes. From the mobility data collected, we gain valuable fundamental understanding of how DNA (both untagged and tagged) migrate within transient micelle networks during electrophoresis. Untagged dsDNA in transient micelle networks follows the general trend of DNA separated in diluted polymer solution similar to a mechanism called transient entanglement coupling. For tagged dsDNA, two distinct mobility regimes are observed: buffer dependent regime for short lengths and obstacle avoidance regime for long lengths. More importantly, we demonstrate that the use of sequence specific alkylated γPNA probes in MTE allows for detection and separation of particular DNA in the presence of other DNA of similar size -- an application suitable for at-line detection of viral/bacterial contaminants in cell culture. Furthermore, we verify that sequence specific γPNAA can also be used to target terminal sequences of digested DNA for the analysis of unknown biologically derived DNA via MTE.