Title page for ETD etd-06072011-220250


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 (ÁCE) 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/cm▓ of total DNA). (2) A continuous-flow polymerase chain reaction (CFPCR) module fabricated in polycarbonate (Tg ~150 ║C) 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) ÁCE module fabricated in poly(methylmethacrylate), which utilized a bioaffinity selection and purification bed (2.9-ÁL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to ÁCE. 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-Ám width; 100-Ám height; total surface area of ~117 mm▓). 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-ÁCE 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 ÁCE, 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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