| Type of Document |
Dissertation |
| Author |
Njoroge, Samuel Kimani
|
| Author's Email Address |
snjoro1@tigers.lsu.edu, sknjoros@gmail.com |
| URN |
etd-06072011-220250 |
| Title |
Integrated Modular Microfliuidic System for Forensic Alu DNA Typing |
| Degree |
Doctor of Philosophy (Ph.D.) |
| Department |
Chemistry |
| Advisory Committee |
| Advisor Name |
Title |
| Soper, Steven A. |
Committee Chair |
| Warner, Isiah M. |
Committee Member |
| Maverick, Andrew W. |
Committee Member |
| Taylor, Carol M. |
Committee Member |
| Pettis, Gregg S. |
Dean's Representative |
|
| Keywords |
- micro capillary electrophoresis
- ethnicity dertemination
- solid-phase extraction
- continuous flow PCR
- DNA analysis
- DNA profile
- integrated microsystems
- microfluidics
- modular systems
- Alu elements
- micro-PCR devices
- gender determination
|
| Date of Defense |
2011-03-09 |
| Availability |
unrestricted |
Abstract
Driven by the numerous applications of genome-related research, fully integrated microfluidic systems have been developed that have advanced the capabilities of molecular and, in particular, genetic analyses. A brief overview on integrated microfluidic systems for DNA analysis is given in Chapter 1 followed by a report on micro-capillary electrophoresis (µCE) of Alu elements with laser-induced fluorescence (LIF) detection, in which the monomorphic Alu insertions on the X and Y chromosomes were utilized to detect male DNA in large female DNA background (Y: X = 1:19) without cell sorting prior to the determination. The polymorphic Alu loci with known restricted geographical distribution were used for ethnicity determination. A valveless integrated microsystem that consists of three modules is discussed as well: (1) A solid-phase extraction (SPE) module microfabricated on polycarbonate, for DNA extraction from whole cell lysates (extraction bed capacity ~209 ±35.6 ng/cm² of total DNA). (2) A continuous-flow polymerase chain reaction (CFPCR) module fabricated in polycarbonate (Tg ~150 ºC) in which selected gene fragments were amplified using biotin and fluorescently-labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones. (3) µCE module fabricated in poly(methylmethacrylate), which utilized a bioaffinity selection and purification bed (2.9-µL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to µCE. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed where they were extracted onto the surface of the poly(methylmethacrylate) micropillars (50-µm width; 100-µm height; total surface area of ~117 mm²). This SPE process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (Kd =10-15M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single stranded DNA released for size-based separations and LIF detection. The multiplexed SPE-CFPCR-µCE yielded detectable fluorescence signal (S/N≥3; LOD ~75 cells) for Alu DNA amplicons for gender and ethnicity determinations with a separation efficiency of ~1.5 x105 plates/m. Compared to traditional cross-T injection procedures typically used for µCE, the affinity preconcentration and injection procedure generated signal enhancements of 17-40 fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation.
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56K Modem |
ISDN (64 Kb) |
ISDN (128 Kb) |
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Njorogecatalogingabstract.pdf |
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Njorogediss.pdf |
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