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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
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?
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
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
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
ME 381DNA Chip TeamDecember 5, 2003
DNA Structure and Nomenclature
• Double Helix
• Four Bases
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.
ME 381DNA Chip TeamDecember 5, 2003
Genes and mRNA in Protein Production
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.
ME 381DNA Chip TeamDecember 5, 2003
Chip Mechanisms
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
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
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
ME 381DNA Chip TeamDecember 5, 2003
Manufacturing Oligonucleotide Arrays
• Masking / DNA Development Process
O O O O O O OH O O OOH OH
O O OT T T T C O CT T
GCT
GGC
TAG
ACC
ATT
CAT
1
5
2
4 6
T O O OT T
3
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 hybridizeInformation from
millions of strands in single dot
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
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
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
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
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
ME 381DNA Chip TeamDecember 5, 2003
•Laser Induced Fluorescence (LIF)
•How is this used in data acquisition
link
Testing with LIF
ME 381DNA Chip TeamDecember 5, 2003
•Laser Induced Fluorescence (LIF)
•How is this used in data acquisition
Read:1. Color2. Intensities
This requires very sophisticated computer analysis
Array Analysis
ME 381DNA Chip TeamDecember 5, 2003
•Electrochemical Sensing
•Why do we need other sensing
Micro scale array Nano scale array
Today Tomorrow
There will be a resolution problem
3 μm 3 μm
Nano-Arrays: The Future of Gene Chips
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
ME 381DNA Chip TeamDecember 5, 2003
•Electrochemical Sensing
•Principle
•Oxidation/Reduction
e-
“Electrons flow from the AuElectrode to intercalated MB+ andThen are accepted by the Fe(CN)6
4-”
E.M. Barton, J.K., N.M. Hill, M.G (1999) NucleicAcid Research 27, 4830.
e-
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Electrochemical Sensing(cont)
ME 381DNA Chip TeamDecember 5, 2003
•Electrochemical Sensing
•Principle
•How is this used in data acquisition
A
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Data Acquisition Methodology
ME 381DNA Chip TeamDecember 5, 2003
•Electrochemical Sensing
•Principle
•How is this used in data acquisition
Voltage Readout
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 canbe measured individually unlike in the LIFapproach. This would in turn reduce the sizeof the chip.
Gold
Benefits of Electrochemical Methods
ME 381DNA Chip TeamDecember 5, 2003
•“Wet” and “Dry” Chip set-up
•Principle
•Combination of Biological and Electrical chips
Proposed Chip Concept
CircuitryA
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Nano DNA Array
ME 381DNA Chip TeamDecember 5, 2003
Thank You For Your Time
DNA Chip Team
Raphael Anstey
Mattheiu Chardon
Travis Harper
Questions?