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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
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DNA Structure and Nomenclature
• Double Helix
• Four Bases
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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
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DNA Chip Team
ME 381
Manufacturing Oligonucleotide Arrays
• Masking / DNA Development Process
2
1
O
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OH OH OH O
4
T
T
T
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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
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G A
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A
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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
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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
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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-
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DNA Chip Team
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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
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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
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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