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DNA Chips: MicroArrays and Emerging Nanotechnologies. ME 381 Final Presentation December 5, 2003 Raphael Anstey Matthieu Chardon Travis Harper. What is a DNA Chip?. Micro-Array containing all the genes (roughly 40,000) in the entire Human Genome (complete Genetic Code).

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dna chips microarrays and emerging nanotechnologies
DNA Chips: MicroArrays and Emerging Nanotechnologies

ME 381

Final Presentation

December 5, 2003

Raphael Anstey

Matthieu Chardon

Travis Harper

what is a dna chip
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.
the power of micro arrays
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.
background on dna
Background on DNA
  • To truly understand Deoxy-RiboNucleic Acid(DNA) chips, we must first understand the elegance and complexity of DNA and genetics.
historical introduction
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.
dna structure and nomenclature
DNA Structure and Nomenclature
  • Double Helix
  • Four Bases
genes and mrna in protein production
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.
applying dna principles to chips
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.
slide11

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:
  • molecular medicine
  • microbial genomics
  • bioarcheology
  • DNA identification
  • bioprocessing
slide12

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
slide13

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

UV Light

Mask

Substrate

slide14

OH

OH

OH

O

O

O

T

T

T

O

O

O

O

O

O

T

T

T

T

T

T

O

C

C

GCT

ATT

CAT

GGC

TAG

ACC

Manufacturing Oligonucleotide Arrays

  • Masking / DNA Development Process

2

1

3

O

O

O

O

O

O

4

5

6

slide15

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

slide16

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
slide17

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
slide18

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
sensing data acquisition
Sensing / Data Acquisition
  • Laser Induced Fluorescence (LIF)
    • Principle:
      • Fluorophores are Tagged on the Target Gene

There are two sorts

colors of dies green

red

laser induced fluorescence
Laser Induced Fluorescence

LASER

  • Laser Induced Fluorescence (LIF)
    • Principle:
      • Shine Laser on the Die

Sense the fluorescent light

emitted by thedie with diode

and analyze data with computers

testing with lif
Testing with LIF
  • Laser Induced Fluorescence (LIF)
    • How is this used in data acquisition

link

array analysis
Array Analysis
  • Laser Induced Fluorescence (LIF)
    • How is this used in data acquisition
  • Read:
  • Color
  • Intensities
  • This requires very sophisticated computer analysis
nano arrays the future of gene chips
Nano-Arrays: The Future of Gene Chips

Nano scale array

  • Electrochemical Sensing
    • Why do we need other sensing

Today

Tomorrow

3 μm

3 μm

Micro scale array

There will be a resolution problem

electrochemical sensing
Electrochemical Sensing
  • Electrochemical Sensing
    • Principle
      • Oxidation/Reduction

Modify a part of the DNA

Methylene Blue (MB+)

Anchor to Substrate to gold electrode

electrochemical sensing cont
Electrochemical Sensing(cont)
  • Electrochemical Sensing
    • Principle
      • Oxidation/Reduction

e-

e-

e-

“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-

data acquisition methodology
Data Acquisition Methodology
  • Electrochemical Sensing
    • Principle
      • How is this used in data acquisition

e-

e-

e-

A

voltage readout
Voltage Readout
  • Electrochemical Sensing
    • Principle
      • How is this used in data acquisition
benefits of electrochemical methods
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

proposed chip concept
Proposed Chip Concept
  • “Wet” and “Dry” Chip set-up
    • Principle
      • Combination of Biological and Electrical chips

e-

e-

Circuitry

e-

A

Nano DNA Array

slide30

Thank You For Your Time

Questions?

DNA Chip Team

Raphael Anstey

Mattheiu Chardon

Travis Harper