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DNA Chips: MicroArrays and Emerging Nanotechnologies ME 381 Final Presentation December 5, 2003 Raphael Anstey Matthieu Chardon Travis Harper December 5, 2003 DNA Chip Team ME 381 What is a DNA Chip? • Micro-Array containing all the genes (roughly 40,000) in the entire Human Genome (complete Genetic Code). • Each known gene or “probe” occupies a particular “spot” on the chip, and varying levels of fluorescent activity show varying levels of gene activity in introduced genetic material. • By introducing these samples or “targets” we can determine which genes are most active for traits, immunities, or any hereditary condition including disease. December 5, 2003 DNA Chip Team ME 381 The Power of Micro-Arrays •Micro-Arrays quickly show the relationships between specific genes and specific traits, diseases and the like. •Thus, we efficiently gain valuable insight into how our genetics specifically affect us. December 5, 2003 DNA Chip Team ME 381 Background on DNA •To truly understand Deoxy-RiboNucleic Acid(DNA) chips, we must first understand the elegance and complexity of DNA and genetics. December 5, 2003 DNA Chip Team ME 381 Historical Introduction • Genetics started in 1866 when a monk named Gregor Mendel discovered biological elements called genes that were responsible the possession and hereditary transfer of a single characteristic. • Genes were linked to DNA, but it took James Watson and Francis Crick deduced the double helix structure of DNA in 1953. • Most recently, the joint venture of the Human Genome Project and a company called Celera published the first draft of the human genome in February 2001. December 5, 2003 DNA Chip Team ME 381 DNA Structure and Nomenclature • Double Helix • Four Bases December 5, 2003 DNA Chip Team ME 381 Genes and mRNA in Protein Production • A gene is a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single mRNA that carries the information needed for constructing a protein. Amazingly only 3% of DNA contains genes, the rest is inactive. • “Messenger” Ribonucleic Acid(mRNA) copies the genetic material off of a DNA strand and transports it form the nucleus to the cytoplasm where Amino Acids are grown into proteins. December 5, 2003 DNA Chip Team ME 381 Genes and mRNA in Protein Production December 5, 2003 DNA Chip Team ME 381 Applying DNA Principles to Chips •Chips are designed to either “sequence” or decode genetic strands, or to find genetic matches. • HYBRIDIZATION • The array provides a medium for matching known and unknown DNA samples based on base-pairing (hybridization) rules. The two strands basically combine automatically if correct matching has occurred. December 5, 2003 DNA Chip Team ME 381 Chip Mechanisms December 5, 2003 DNA Chip Team ME 381 The Human Genome • Intended to produce a DNA sequence representing the functional blueprint and evolutionary history of the human species • Identify all of the approximately 30,000 genes in human DNA • Determine sequences of 3 billion chemical base pairs that make up DNA • Expensive arduous process - Eleven years, three billion dollars • Applications in diverse biological fields: o molecular medicine o DNA identification o microbial genomics o bioprocessing o bioarcheology December 5, 2003 DNA Chip Team ME 381 Functional Genomics • Thousands of genes and their products in a given living organism function in a complicated and orchestrated way that creates the mystery of life • Whole picture of gene function is hard to obtain in varying one gene per experiment • Simultaneously analyzing expression levels of a large number of genes provides the opportunity to study the activity of an entire genome • The DNA Chip permits these kinds of analyses December 5, 2003 DNA Chip Team ME 381 Manufacturing Oligonucleotide Arrays • MEMS processing technologies • Photolithography removes DNA terminators • Nucleotide adds itself to exposed strand • DNA is constructed in situ UV Light • Process requires several masking steps Mask Substrate December 5, 2003 DNA Chip Team ME 381 Manufacturing Oligonucleotide Arrays • Masking / DNA Development Process 2 1 O O O O O O OH OH OH O 4 T T T December 5, 2003 O 3 O O T T T O 5 O O T T T C O O 6 O DNA Chip Team C G C T A T T C A T G T G A C G A C C ME 381 Array Hybridization • Single strand oligonucleotides stand on the chip • Hybridization occurs in complementary strands • Each microarray dot contains millions of identical strands Single strands in the area of a microarray dot Strands hybridize Noncomplementary strands in other regions of the chip do not hybridize December 5, 2003 DNA Chip Team Information from millions of strands in single dot ME 381 Scaling Considerations • Desire for high density of experiments • Sample availability limitations • Extremely beneficial to bring DNA Chip analyses to nanoscale • Requires lithography technique with high resolution • Solution found in working with the atomic force microscope December 5, 2003 DNA Chip Team ME 381 Dip Pen Nanolithography • Revolutionary science developed at Northwestern • Allows for deposition of inks, including DNA, at nanometer resolution • Spot sized reduced from 20-40 μm to 50 nm • 100,000 spots can be prepared in area conventionally housing a single spot • Ultra-high-density gene chips • Direct write of DNA onto substrate December 5, 2003 DNA Chip Team ME 381 DPN Parallel Writing • Use of cantilever arrays consisting of multiple pens transforms DPN into a parallel writing tool • Time efficient method to directly deposit DNA onto a substrate December 5, 2003 DNA Chip Team ME 381 Sensing / Data Acquisition •Laser Induced Fluorescence (LIF) •Principle: •Fluorophores are Tagged on the Target Gene There are two sorts colors of dies green red December 5, 2003 DNA Chip Team ME 381 Laser Induced Fluorescence •Laser Induced Fluorescence (LIF) •Principle: •Shine Laser on the Die LASER Sense the fluorescent light emitted by thedie with diode and analyze data with computers December 5, 2003 DNA Chip Team ME 381 Testing with LIF •Laser Induced Fluorescence (LIF) •How is this used in data acquisition link December 5, 2003 DNA Chip Team ME 381 Array Analysis •Laser Induced Fluorescence (LIF) •How is this used in data acquisition Read: 1. Color 2. Intensities This requires very sophisticated computer analysis December 5, 2003 DNA Chip Team ME 381 Nano-Arrays: The Future of Gene Chips •Electrochemical Sensing •Why do we need other sensing Today 3 μm Tomorrow 3 μm Micro scale array Nano scale array There will be a resolution problem December 5, 2003 DNA Chip Team ME 381 Electrochemical Sensing •Electrochemical Sensing •Principle •Oxidation/Reduction Modify a part of the DNA Methylene Blue (MB+) Anchor to Substrate to gold electrode December 5, 2003 DNA Chip Team ME 381 Electrochemical Sensing(cont) •Electrochemical Sensing •Principle •Oxidation/Reduction eee- “Electrons flow from the Au Electrode to intercalated MB+ and Then are accepted by the Fe(CN)64-” E.M. Barton, J.K., N.M. Hill, M.G (1999) Nucleic Acid Research 27, 4830. e- December 5, 2003 DNA Chip Team ME 381 Data Acquisition Methodology •Electrochemical Sensing •Principle •How is this used in data acquisition eee- A December 5, 2003 DNA Chip Team ME 381 Voltage Readout •Electrochemical Sensing •Principle •How is this used in data acquisition December 5, 2003 DNA Chip Team ME 381 Benefits of Electrochemical Methods •Electrochemical Sensing •Principle •Variations/Benefits Ir(bpy)(phen)(phi)3+ Both strands have to be modified when using methylene. It is possible to use other molecules to act as catalyst such as Ir… This is a benefit to because each gene can be measured individually unlike in the LIF approach. This would in turn reduce the size of the chip. Gold December 5, 2003 DNA Chip Team ME 381 Proposed Chip Concept •“Wet” and “Dry” Chip set-up •Principle •Combination of Biological and Electrical chips eee- Circuitry Nano DNA Array December 5, 2003 DNA Chip Team A ME 381 Thank You For Your Time Questions? DNA Chip Team Raphael Anstey Mattheiu Chardon Travis Harper December 5, 2003 DNA Chip Team ME 381