Ebook Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (Nir-Lif) And Single Molecule Detection In Microfluidic Devices
In June 2000, a rough draft of the human genome was completed. The draft sequence provided a scaffold of sequences across 90% of the human genome. Some of the main goals of the project were to identify the entire 30,000 genes in the human genome, determine the sequence of all the 3 billion chemical bases that make up the human genome, and store this information in a database. Due to this an avalanche of genome data has been produced.
This brings new challenges to biological studies in transcription, proteomics, structural genomics, experimental methodologies and comparative genomics. Conversely, deciphering the genome has allowed development of new technologies for diagnosing and treating human disease through mutation or polymorphism analysis of disease-causing genes.
CONTENTS
ACKNOWLEDGMENTS
LIST OF TABLES
LIST OF FIGURES
COMMON ABBREVIATIONS AND ACRONYMS
ABSTRACT
INTRODUCTION
- I.1. Overview of the Human Genome Project
I.2. Genetic Variations and Diseases
I.3. Structure of DNA
I.4. Mutation Detection
I.5. Research Focus
I.6. References
CHAPTER 1. GENOMIC ANALYSIS
- 1.1. Genome Analysis
1.2. Mutation Detection Techniques
1.3. References
CHAPTER 2. PCR AMPLIFICATION AND SEQUENCING OF SINGLE COPY DNA MOLECULES
- 2.1. Introduction
2.2. PCR Amplification in Genome Analysis
2.3. Classification of DNA Polymerase
2.4. PCR Amplification Methods
2.5. DNA Sequencing Using Chain Terminating Inhibitor (Sanger Dideoxy Method)
2.6. Current Research in Single Copy Amplification of DNA Molecules
2.7. Experimental Section
- 2.7.1 Polymerase Chain Reactions
2.7.2. Capillary Electrophoresis
2.7.3. DNA Sequencing
2.8. Results and Discussion
2.9. Conclusions
2.10. References
CHAPTER 3. SINGLE MOLECULE DETECTION OF DOUBLE-STRANDED DNA IN POLY(METHYLMETHACRYLATE) AND POLYCARBONATE MICROFLUIDIC DEVICES
- 3.1. Overview of Single Molecule Detection
3.2. Basic Principles of SMD
3.3. Single Molecule Detection in Solution
3.4. Application of SMD in Solution
3.5. Single Molecule Detection of Biomolecules
3.6. Single Molecule Detection of Genetic Assays
3.7. Microfluidic Devices
- 3.7.1. Microfabrication Techniques
3.7.2. Hot-Embossing
3.7.3. LIGA
3.7.3 Electrokinetic Pumping in Polymers Devices
3.7.4 Applications of Polymer Microfluidic Devices
3.8. Experimental Section
- 3.8.1. Microchip Fabrication
3.8.2. Materials and Reagents
3.8.3. DNA Labeling
3.8.4. Instrumentation
3.8.5. Electrokinetic Pumping and Data Acquisition
3.8.6. Data Analysis
3.9. Results and Discussion
3.10. Conclusions
3.11. References
CHAPTER 4. APPROACHING REAL TIME MOLECULAR DIAGNOSTICS: SINGLE-PAIR FLUORESCENCE RESONANCE ENERGY TRANSFER DETECTION FOR THE ANALYSIS OF LOW ABUNDANT POINT MUTATIONS IN K-RAS ONCOGENES
- 4.1. Introduction
4.2. Colorectal Cancer
4.3. Ligase Detection Reaction (LDR) for Colorectal Cancer
4.4. DNA Ligase Reaction Mechanism
4.5. Fluorescence Resonance Energy Transfer (FRET) Detection
- 4.5.1. Theory of FRET
4.5.2. FRET Conditions
4.5.3 Applications of FRET
4.6. Molecular Beacon Technology
- 4.6.1 Design of Molecular Beacons
4.7. Experimental Section
- 4.7.1. Genomic DNA Extraction from Cell Lines
4.7.2. PCR Amplification of Genomic DNA
4.7.3. Microfluidic Devices
4.7.4. Ligation Primer and DNA Hairpins
4.7.5. Ligation Protocol
4.7.6. Instrumentation
4.7.7. Electrokinetic Pumping
4.7.8. Data Analysis
4.8. Results and Discussion
- 4.8.1. DNA Hairpin Studies
4.8.2. Single-pair FRET Detection
4.9. Conclusions
4.10. References
CHAPTER 5. CONCLUSION AND FUTURE WORK
- 5.1. Future Work
VITA
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