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Author: Samantha Anne Cronier Publisher: ISBN: Category : Languages : en Pages : 110
Book Description
Recent work in the areas of microfluidic technology and innovative new chemistries have made possible tremendous improvements in our ability to determine the nucleotide sequence of DNA. These advances are already changing the way doctors diagnose and treat human disease and enabling scientists to undertake genetic studies never before possible. In this dissertation I provide a brief history of DNA sequencing methods as well as a general description of microfluidic technologies, in particular those used for genetic analysis. Next, I describe my dissertation research on the development of a highly efficient fully integrated microfluidic platform for Sanger DNA sequencing, including automated thermal cycling, purification, concentration and in-line injection of the extension fragments for microchip capillary electrophoresis separation. The two-layer glass device that I developed features two independently operated valve-free systems, comprised of a 200 nL thermal cycling reactor with resistive temperature detector, a 1.2 nL in situ photopolymerized oligonucleotide affinity capture gel for post reaction clean-up and inline injection, and an 18-cm long capillary electrophoresis channel for separation. Integration of the efficient photopolymerized affinity gel capture allows sequencing from only 350 - 500 attomoles of starting DNA template. Using this device, I was able to sequence 507 ± 31 bases at 99% accuracy. In addition, I show that this method is compatible with single cell genetic analysis techniques (SCGA) by sequencing from the small amounts (1̃00 attomoles) of amplified DNA bound to agarose microbeads that can be produced from single cells. This dissertation concludes with a discussion of the future of DNA sequencing and the feasibility of performing single cell DNA sequencing using the Microbead Integrated DNA Sequencing (MINDS) method.
Author: Samantha Anne Cronier Publisher: ISBN: Category : Languages : en Pages : 110
Book Description
Recent work in the areas of microfluidic technology and innovative new chemistries have made possible tremendous improvements in our ability to determine the nucleotide sequence of DNA. These advances are already changing the way doctors diagnose and treat human disease and enabling scientists to undertake genetic studies never before possible. In this dissertation I provide a brief history of DNA sequencing methods as well as a general description of microfluidic technologies, in particular those used for genetic analysis. Next, I describe my dissertation research on the development of a highly efficient fully integrated microfluidic platform for Sanger DNA sequencing, including automated thermal cycling, purification, concentration and in-line injection of the extension fragments for microchip capillary electrophoresis separation. The two-layer glass device that I developed features two independently operated valve-free systems, comprised of a 200 nL thermal cycling reactor with resistive temperature detector, a 1.2 nL in situ photopolymerized oligonucleotide affinity capture gel for post reaction clean-up and inline injection, and an 18-cm long capillary electrophoresis channel for separation. Integration of the efficient photopolymerized affinity gel capture allows sequencing from only 350 - 500 attomoles of starting DNA template. Using this device, I was able to sequence 507 ± 31 bases at 99% accuracy. In addition, I show that this method is compatible with single cell genetic analysis techniques (SCGA) by sequencing from the small amounts (1̃00 attomoles) of amplified DNA bound to agarose microbeads that can be produced from single cells. This dissertation concludes with a discussion of the future of DNA sequencing and the feasibility of performing single cell DNA sequencing using the Microbead Integrated DNA Sequencing (MINDS) method.
Author: Samuel Njoroge Publisher: LAP Lambert Academic Publishing ISBN: 9783846539866 Category : Languages : en Pages : 232
Book Description
To produce a microsystem with favorable performance characteristics for genetic-based analyses, several key operational elements must be strategically chosen, including device substrate material, temperature control, fluidic control and reaction product readout. The advantages afforded by fully integrated microfluidic systems to enable challenging applications, such as single-copy DNA sequencing, single-cell gene expression analysis, pathogen detection, and forensic DNA analysis in formats that provide high throughput and point-of-analysis capabilities must be addressed by the researcher. This book focus on discussion on microfluidic systems that are composed of two or more microdevices directed toward DNA analyses. The discussions primarily focus on the integration of various processing steps with u-capillary electrophoresis (uCE) with discoveries that have led to the development of fully autonomous and functional integrated systems for genome processing that can supply "sample in / answer out" capabilities."
Author: Kirsty Jane Shaw Publisher: ISBN: Category : Languages : en Pages :
Book Description
An evaluation of DNA extraction and amplification performed in microfluidic systems was carried out, with the aim of integrating the two processes in a single microfluidic device. This integrated device will then be incorporated upstream of capillary gel electrophoresis and fluorescence-based detection for development of a completely integrated genetic analysis system. DNA extraction was performed using a silica substrate with both hydrodynamic and electro-osmotic pumping (EOP), resulting in maximum DNA extraction efficiencies of 82% and 52% respectively under optimised conditions. While the DNA extraction efficiency was lower using EOP, this method eliminates the need for external pumps and ensures easier mechanical connection to the microfluidic device. The use of thermally activated silica monoliths as the solid-phase resulted in superior DNA extraction efficiencies compared to when photo-initiated monoliths and silica beads were used. DNA amplification of up to nine forensically relevant loci was successfully achieved on the microfluidic device in volumes as low as 1.1 microlitres using Peltier heating. A combination of silanisation and dynamic passivation was required to prevent PCR inhibition resulting from DNA polymerase adsorption. A custom-built microwave heating system was also evaluated, which was capable of heating and cooling rates of 65°C/second and 58°C/second, respectively. EOP was used in the generation of an integrated microfluidic device, for DNA extraction and amplification. The silica monolith used as the solid-phase for DNA extraction also acted as a pump for electrokinetic movement. All necessary reagents for carrying out both DNA extraction and amplification were encapsulated in agarose gel and pre-loaded onto the microfluidic device creating a self-contained, ready-to-use system. Following addition of the biological sample to the microfluidic device, all electrokinetic movement and thermal cycling was controlled using a custom-built operating system.
Author: Nicholas Michael Toriello Publisher: ISBN: 9780549834182 Category : Languages : en Pages : 438
Book Description
Next, I developed a microdevice for RNA analysis that integrates 1-step reverse transcription and 30 cycles of PCR (RT-PCR) amplification with capillary electrophoresis (CE) separation and fluorescence detection of the amplicons. The system demonstrates attomolar detection sensitivity (
Author: Xiujun (James) Li Publisher: Newnes ISBN: 0444594612 Category : Science Languages : en Pages : 486
Book Description
Multidisciplinary Microfluidic and Nanofluidic Lab-on-a-Chip: Principles and Applications provides chemists, biophysicists, engineers, life scientists, biotechnologists, and pharmaceutical scientists with the principles behind the design, manufacture, and testing of life sciences microfluidic systems. This book serves as a reference for technologies and applications in multidisciplinary areas, with an emphasis on quickly developing or new emerging areas, including digital microfluidics, nanofluidics, papers-based microfluidics, and cell biology. The book offers practical guidance on how to design, analyze, fabricate, and test microfluidic devices and systems for a wide variety of applications including separations, disease detection, cellular analysis, DNA analysis, proteomics, and drug delivery. Calculations, solved problems, data tables, and design rules are provided to help researchers understand microfluidic basic theory and principles and apply this knowledge to their own unique designs. Recent advances in microfluidics and microsystems for life sciences are impacting chemistry, biophysics, molecular, cell biology, and medicine for applications that include DNA analysis, drug discovery, disease research, and biofluid and environmental monitoring. - Provides calculations, solved problems, data tables and design rules to help understand microfluidic basic theory and principles - Gives an applied understanding of the principles behind the design, manufacture, and testing of microfluidic systems - Emphasizes on quickly developing and emerging areas, including digital microfluidics, nanofluidics, papers-based microfluidics, and cell biology
Author: Wei-Cheng Tian Publisher: Springer Science & Business Media ISBN: 0387094806 Category : Technology & Engineering Languages : en Pages : 429
Book Description
Microfluidics for Biological Applications provides researchers and scientists in the biotechnology, pharmaceutical, and life science industries with an introduction to the basics of microfluidics and also discusses how to link these technologies to various biological applications at the industrial and academic level. Readers will gain insight into a wide variety of biological applications for microfluidics. The material presented here is divided into four parts, Part I gives perspective on the history and development of microfluidic technologies, Part II presents overviews on how microfluidic systems have been used to study and manipulate specific classes of components, Part III focuses on specific biological applications of microfluidics: biodefense, diagnostics, high throughput screening, and tissue engineering and finally Part IV concludes with a discussion of emerging trends in the microfluidics field and the current challenges to the growth and continuing success of the field.
Author: Samantha Anne Cronier
Author: Samantha Anne Cronier
Author: Golnaz Vahedi
Author: Samuel Njoroge
Author: Christopher John Easley
Author: Robert Glenn Blazej
Author: Kirsty Jane Shaw
Author: Joan Marie Bienvenue
Author: Nicholas Michael Toriello
Author: Xiujun (James) Li
Author: Wei-Cheng Tian