DNA Chip


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DNA Chip

  1. 1. DNA Chips: MicroArrays and Emerging Nanotechnologies ME 381 Final Presentation December 5, 2003 Raphael Anstey Matthieu Chardon Travis Harper
  2. 2. What is a DNA Chip? <ul><li>Micro-Array containing all the genes (roughly 40,000) in the entire Human Genome (complete Genetic Code). </li></ul><ul><li>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. </li></ul><ul><li>By introducing these samples or “targets” we can determine which genes are most active for traits, immunities, or any hereditary condition including disease. </li></ul>
  3. 3. The Power of Micro-Arrays <ul><li>Micro-Arrays quickly show the relationships between specific genes and specific traits, diseases and the like. </li></ul><ul><li>Thus, we efficiently gain valuable insight into how our genetics specifically affect us. </li></ul>
  4. 4. Background on DNA <ul><li>To truly understand Deoxy-RiboNucleic Acid(DNA) chips, we must first understand the elegance and complexity of DNA and genetics. </li></ul>                                               
  5. 5. Historical Introduction <ul><li>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. </li></ul><ul><li>Genes were linked to DNA, but it took James Watson and Francis Crick deduced the double helix structure of DNA in 1953. </li></ul><ul><li>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. </li></ul>
  6. 6. DNA Structure and Nomenclature <ul><li>Double Helix </li></ul><ul><li>Four Bases </li></ul>
  7. 7. Genes and mRNA in Protein Production <ul><li>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. </li></ul><ul><li>“ 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. </li></ul>
  8. 8. Genes and mRNA in Protein Production
  9. 9. Applying DNA Principles to Chips <ul><li>Chips are designed to either “sequence” or decode genetic strands, or to find genetic matches. </li></ul><ul><li>HYBRIDIZATION </li></ul><ul><ul><li>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. </li></ul></ul>
  10. 10. Chip Mechanisms
  11. 11. The Human Genome <ul><li>Intended to produce a DNA sequence representing the functional blueprint and evolutionary history of the human species </li></ul><ul><li>Identify all of the approximately 30,000 genes in human DNA </li></ul><ul><li>Determine sequences of 3 billion chemical base pairs that make up DNA </li></ul><ul><li>Expensive arduous process - Eleven years, three billion dollars </li></ul><ul><li>Applications in diverse biological fields: </li></ul><ul><li>molecular medicine </li></ul><ul><li>microbial genomics </li></ul><ul><li>bioarcheology </li></ul><ul><li>DNA identification </li></ul><ul><li>bioprocessing </li></ul>
  12. 12. Functional Genomics <ul><li>Thousands of genes and their products in a given living organism function in a complicated and orchestrated way that creates the mystery of life </li></ul><ul><li>Whole picture of gene function is hard to obtain in varying one gene per experiment </li></ul><ul><li>Simultaneously analyzing expression levels of a large number of genes provides the opportunity to study the activity of an entire genome </li></ul><ul><li>The DNA Chip permits these kinds of analyses </li></ul>
  13. 13. Manufacturing Oligonucleotide Arrays <ul><li>MEMS processing technologies </li></ul><ul><li>Photolithography removes DNA terminators </li></ul><ul><li>Nucleotide adds itself to exposed strand </li></ul><ul><li>DNA is constructed in situ </li></ul><ul><li>Process requires several masking steps </li></ul>Substrate Mask UV Light
  14. 14. Manufacturing Oligonucleotide Arrays <ul><li>Masking / DNA Development Process </li></ul>O O O O O O 1 5 2 4 6 3 OH O O O OH OH O O O T T T T C O C T T GCT GGC TAG ACC ATT CAT T O O O T T
  15. 15. Array Hybridization <ul><li>Single strand oligonucleotides stand on the chip </li></ul><ul><li>Hybridization occurs in complementary strands </li></ul><ul><li>Each microarray dot contains millions of identical strands </li></ul>Single strands in the area of a microarray dot Strands hybridize Noncomplementary strands in other regions of the chip do not hybridize Information from millions of strands in single dot
  16. 16. Scaling Considerations <ul><li>Desire for high density of experiments </li></ul><ul><li>Sample availability limitations </li></ul><ul><li>Extremely beneficial to bring DNA Chip analyses to nanoscale </li></ul><ul><li>Requires lithography technique with high resolution </li></ul><ul><li>Solution found in working with the atomic force microscope </li></ul>
  17. 17. Dip Pen Nanolithography <ul><li>Revolutionary science developed at Northwestern </li></ul><ul><li>Allows for deposition of inks, including DNA, at nanometer resolution </li></ul><ul><li>Spot sized reduced from 20-40 μm to 50 nm </li></ul><ul><li>100,000 spots can be prepared in area conventionally housing a single spot </li></ul><ul><li>Ultra-high-density gene chips </li></ul><ul><li>Direct write of DNA onto substrate </li></ul>
  18. 18. DPN Parallel Writing <ul><li>Use of cantilever arrays consisting of multiple pens transforms DPN into a parallel writing tool </li></ul><ul><li>Time efficient method to directly deposit DNA onto a substrate </li></ul>
  19. 19. Sensing / Data Acquisition <ul><li>Laser Induced Fluorescence (LIF) </li></ul><ul><ul><li>Principle: </li></ul></ul><ul><ul><ul><li>Fluorophores are Tagged on the Target Gene </li></ul></ul></ul>There are two sorts colors of dies green red
  20. 20. Laser Induced Fluorescence <ul><li>Laser Induced Fluorescence (LIF) </li></ul><ul><ul><li>Principle: </li></ul></ul><ul><ul><ul><li>Shine Laser on the Die </li></ul></ul></ul>Sense the fluorescent light emitted by thedie with diode and analyze data with computers LASER
  21. 21. Testing with LIF <ul><li>Laser Induced Fluorescence (LIF) </li></ul><ul><ul><li>How is this used in data acquisition </li></ul></ul>link
  22. 22. Array Analysis <ul><li>Laser Induced Fluorescence (LIF) </li></ul><ul><ul><li>How is this used in data acquisition </li></ul></ul><ul><li>Read: </li></ul><ul><li>Color </li></ul><ul><li>Intensities </li></ul><ul><li>This requires very sophisticated computer analysis </li></ul>
  23. 23. Nano-Arrays: The Future of Gene Chips <ul><li>Electrochemical Sensing </li></ul><ul><ul><li>Why do we need other sensing </li></ul></ul>Micro scale array Today Tomorrow There will be a resolution problem 3 μ m 3 μ m Nano scale array
  24. 24. Electrochemical Sensing <ul><li>Electrochemical Sensing </li></ul><ul><ul><li>Principle </li></ul></ul><ul><ul><ul><li>Oxidation/Reduction </li></ul></ul></ul>Methylene Blue (MB + ) Anchor to Substrate to gold electrode Modify a part of the DNA
  25. 25. Electrochemical Sensing(cont) <ul><li>Electrochemical Sensing </li></ul><ul><ul><li>Principle </li></ul></ul><ul><ul><ul><li>Oxidation/Reduction </li></ul></ul></ul>e - “ Electrons flow from the Au Electrode to intercalated MB + and Then are accepted by the Fe(CN) 6 4- ” E.M. Barton, J.K., N.M. Hill, M.G (1999) Nucleic Acid Research 27, 4830. e - e - e -
  26. 26. Data Acquisition Methodology <ul><li>Electrochemical Sensing </li></ul><ul><ul><li>Principle </li></ul></ul><ul><ul><ul><li>How is this used in data acquisition </li></ul></ul></ul>A e - e - e -
  27. 27. Voltage Readout <ul><li>Electrochemical Sensing </li></ul><ul><ul><li>Principle </li></ul></ul><ul><ul><ul><li>How is this used in data acquisition </li></ul></ul></ul>
  28. 28. Benefits of Electrochemical Methods <ul><li>Electrochemical Sensing </li></ul><ul><ul><li>Principle </li></ul></ul><ul><ul><ul><li>Variations/Benefits </li></ul></ul></ul>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
  29. 29. Proposed Chip Concept <ul><li>“ Wet” and “Dry” Chip set-up </li></ul><ul><ul><li>Principle </li></ul></ul><ul><ul><ul><li>Combination of Biological and Electrical chips </li></ul></ul></ul>Circuitry A e - e - e - Nano DNA Array
  30. 30. Thank You For Your Time DNA Chip Team Raphael Anstey Mattheiu Chardon Travis Harper Questions?