Cationic, random-coil protein polymers for free-solution conjugate electrophoresis (FSCE) in microchannels
Abstract/Contents
- Abstract
- Access to an individual's genetic data, often for a small set of genes, will offer increasing value in medical practice; e.g., in cancer treatment it may be useful to track sequence alterations in oncogenes over time, or even spatially within a tumor. Such applications create a need for rapid, low-cost, scalable technologies for high-accuracy sequencing of human DNA. Some regions of the human genome, particularly repeat-rich loci, are challenging to analyze accurately with short-read, next-gen DNA sequencing technologies. Long-read, high-accuracy sequencing is done now by capillary array electrophoresis (CE), but DNA separations on microdevices are at least 10 times faster and deliver equally long, accurate reads. Free-Solution Conjugate Electrophoresis (FSCE) is a novel method of DNA sequencing ideal for implementation on microfluidic devices, as it obviates the need for viscous polymer solutions to separate DNA. FSCE uses a monodisperse protein "drag-tag", tethered end-on to one terminus of ssDNA fragments, to enable sequencing in free solution. The most difficult aspect of developing FSCE is the creation of long (> 500-amino acid (aa)), totally monodisperse protein drag-tags needed for long reads. Recent studies in FSCE indicated that introducing positive charges in a drag-tag would help to achieve higher hydrodynamic drag without requirements of significantly increasing the size of the drag-tag. In this dissertation, a new family of de novo-designed proteins with intentionally included cationic (Arg) amino acids was designed. A faster and easier purification method combining affinity chromatography and tag-removal was established to create protein polymers with improved monodispersity required for FSCE applications. After rigorous characterization, these novel protein drag-tags were analyzed by free-solution separations and FSCE sequencing. Through these studies, I investigated critical biophysical attributes of positively charged drag-tags important to FSCE performance and identified the limits for inclusion of positive charges in sequences of protein polymer drag-tags for successful FSCE applications. The investigations of two post-Sanger conjugation strategies were also initiated and were proven to enable the utilization of previously unusable larger/higher charged protein drag-tags that suppressed the enzymatic extension when attached to sequencing primers prior to the Sanger reaction.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2011 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Wang, Xiaoxiao |
|---|---|
| Associated with | Stanford University, Department of Bioengineering. |
| Primary advisor | Barron, Annelise E |
| Thesis advisor | Barron, Annelise E |
| Thesis advisor | Heilshorn, Sarah |
| Thesis advisor | Swartz, James R |
| Advisor | Heilshorn, Sarah |
| Advisor | Swartz, James R |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Xiaoxiao Wang. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2011. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2011 by Xiaoxiao Wang
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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