Computational Biology and Bioinformatics Lab. Songhwan Hwang Functional Genomics DNA Microarray Technology

Introduction Introduction Microarray design and probe preparation Microarray fabrication Sample preparation and labeling with fluoresceins Microarray hybridization Microarray scanning Data mining and analysis Microarrays (or microchips) are a powerful genomic technology  Research questions - Gene expression, community analysis and genotyping

Types of microarrays and advantages Types of microarrays and advantages  Microarray types - DNA microarrays - Oligonucleotide microarrays  Advantages of microarrays - High-throughput and parallel analysis - High sensitivity - Differential display - Low background signal noise - Real-time data analysis - Automation Light-directed or standard phosphoramidite chmistries Chemical attachment of prefabricated oligonucleotides to solid supports

Microarray fabrication Microarray fabrication  Microarray fabrication substrates and modification - Substrates : nonporous substrate, porous substrate - Attachment strategies : electrostatic interaction, covalent bonding - Surface modification : silanization, dendrimeric linker coating, gel coating, nitrocellulose coating

Microarray fabrication Microarray fabrication  Critical issues for microarray fabrication - Microarray density : spot size, pin configuration - Reproducibility - Storage time - Contamination - Evaluation of printing quality  Arraying technology - Light-directed synthesis - Contact printing : solid pins, split pins, pin and ring - Noncontact ink-jet printing : piezoelectric pumps, syringe-solenoid printing technology

Microarray hybridization and detection Microarray hybridization and detection  Probe design, target preparation, and quality - Probe design and synthesis - Characteristics of fluorescence - Target labeling and quality : direct labeling, indirect labeling

Microarray hybridization and detection Microarray hybridization and detection  Critical issues for hybridization and detection - Probe DNA retention and quantitative hybridization - Target labeling and availability - Spatial resolution and cross-talk - Photobleaching and scanning parameters  Hybridization - Before hybridization, the free functional groups on the slides can be washed away. - After prehybridization, the microarray is hybridized with fluorescently labeled target DNA or RNA for a period of time.  Detection - The confocal scanning microscope and CCD camera have been used successfully for microarray detection.

Microarray image processing Microarray image processing  Data acquisition - Location of spots : center - Spot size and shape : circular and homogeneous - Location of the grids on the images : fixed - Slides : no contamination - Background intensity : very low and uniform  Assessment of spot quality and reliability, and background subtraction - Identification of poor slides - Identification of poor-quality spots - Removal of outlying spots  Background subtraction - Background subtraction is necessary to distinguish signals from noise and allows the comparison of specific signals.

 General approaches to revealing differences in gene expression Using microarrays to monitor gene expression Using microarrays to monitor gene expression

Summary Summary  Microarrays have had a great impact on many areas of research, including medical science, agriculture, industry, and environmental studies.  Microarray fabrication is one of the key steps of microarray technology. - The substrate used for printing microarrays has a significant impact on the overall microarray experiment.  Probe design and synthesis are critical steps in generating high-quality microarrays for analysis.  Image processing is an important step for microarray-based analysis.  Whole-genome microarray is a powerful approach to studying gene expression.

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