1. ME 381DNA Chip TeamDecember 5, 2003 DNA Chips: MicroArrays and Emerging Nanotechnologies ME 381 Final Presentation December 5, 2003 Raphael Anstey Matthieu Chardon Travis Harper

2. ME 381DNA Chip TeamDecember 5, 2003 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. What is a DNA Chip?

3. ME 381DNA Chip TeamDecember 5, 2003 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. The Power of Micro-Arrays

4. ME 381DNA Chip TeamDecember 5, 2003 To truly understand Deoxy-RiboNucleic Acid(DNA) chips, we must first understand the elegance and complexity of DNA and genetics. Background on DNA

5. ME 381DNA Chip TeamDecember 5, 2003 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. Historical Introduction

6. ME 381DNA Chip TeamDecember 5, 2003 DNA Structure and Nomenclature Double Helix Four Bases

7. ME 381DNA Chip TeamDecember 5, 2003 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.

8. ME 381DNA Chip TeamDecember 5, 2003 Genes and mRNA in Protein Production

9. ME 381DNA Chip TeamDecember 5, 2003 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.

10. ME 381DNA Chip TeamDecember 5, 2003 Chip Mechanisms

11. ME 381DNA Chip TeamDecember 5, 2003 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 microbial genomics o bioarcheology o DNA identification o bioprocessing

12. ME 381DNA Chip TeamDecember 5, 2003 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

13. ME 381DNA Chip TeamDecember 5, 2003 Manufacturing Oligonucleotide Arrays MEMS processing technologies Photolithography removes DNA terminators Nucleotide adds itself to exposed strand DNA is constructed in situ Process requires several masking steps Substrate Mask UV Light

14. ME 381DNA Chip TeamDecember 5, 2003 Manufacturing Oligonucleotide Arrays Masking / DNA Development Process OOO O O O OH O O O O O O TT T T C O C TT GCTGCT GGCGGC TAGTAG ACCACC ATTATT CATCAT 1 5 2 4 6 T O O O TT 3

15. ME 381DNA Chip TeamDecember 5, 2003 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 Information from millions of strands in single dot

16. ME 381DNA Chip TeamDecember 5, 2003 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

17. ME 381DNA Chip TeamDecember 5, 2003 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

18. ME 381DNA Chip TeamDecember 5, 2003 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

19. ME 381DNA Chip TeamDecember 5, 2003 Laser Induced Fluorescence (LIF) Principle: Fluorophores are Tagged on the Target Gene There are two sorts colors of dies green red Sensing / Data Acquisition

20. ME 381DNA Chip TeamDecember 5, 2003 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 Laser Induced Fluorescence

21. ME 381DNA Chip TeamDecember 5, 2003 Laser Induced Fluorescence (LIF) How is this used in data acquisition link Testing with LIF

22. ME 381DNA Chip TeamDecember 5, 2003 Laser Induced Fluorescence (LIF) How is this used in data acquisition Read: 1.Color 2.Intensities This requires very sophisticated computer analysis Array Analysis

23. ME 381DNA Chip TeamDecember 5, 2003 Electrochemical Sensing Why do we need other sensing Micro scale array Nano scale array TodayTomorrow There will be a resolution problem 3 μm Nano-Arrays: The Future of Gene Chips

24. ME 381DNA Chip TeamDecember 5, 2003 Electrochemical Sensing Principle Oxidation/Reduction Methylene Blue (MB + ) Anchor to Substrate to gold electrode Modify a part of the DNA Electrochemical Sensing

25. ME 381DNA Chip TeamDecember 5, 2003 Electrochemical Sensing Principle Oxidation/Reduction e-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-e- e-e- Electrochemical Sensing(cont)

26. ME 381DNA Chip TeamDecember 5, 2003 Electrochemical Sensing Principle How is this used in data acquisition A e-e- e-e- e-e- Data Acquisition Methodology

27. ME 381DNA Chip TeamDecember 5, 2003 Electrochemical Sensing Principle How is this used in data acquisition Voltage Readout

28. ME 381DNA Chip TeamDecember 5, 2003 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 Benefits of Electrochemical Methods

29. ME 381DNA Chip TeamDecember 5, 2003 “Wet” and “Dry” Chip set-up Principle Combination of Biological and Electrical chips Proposed Chip Concept Circuitry A e-e- e-e- e-e- Nano DNA Array

30. ME 381DNA Chip TeamDecember 5, 2003 Thank You For Your Time DNA Chip Team Raphael Anstey Mattheiu Chardon Travis Harper Questions?