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

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


• 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?


•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


•To truly understand Deoxy-RiboNucleic Acid(DNA) chips, we must first understand the elegance and complexity of DNA and genetics.

Background on DNA


• 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


DNA Structure and Nomenclature

• Double Helix

• Four Bases


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.


Genes and mRNA in Protein Production


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.


Chip Mechanisms


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


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


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


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


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


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


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


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


•Laser Induced Fluorescence (LIF)

•Principle:

•Fluorophores are Tagged on the Target Gene

There are two sorts colors of dies green red

Sensing / Data Acquisition



•Principle:

•Shine Laser on the Die

LASER

Sense the fluorescent light emitted by thedie with diode and analyze data with computers

Laser Induced Fluorescence



•How is this used in data acquisition

link

Testing with LIF




Read:1. Color2. Intensities

This requires very sophisticated computer analysis

Array Analysis


•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



•Principle

•Oxidation/Reduction

Methylene Blue (MB+)

Anchor to Substrate to gold electrode

Modify a part of the DNA

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-

e-

e-

Electrochemical Sensing(cont)



•Principle


A

e-

e-

e-

Data Acquisition Methodology



•Principle


Voltage Readout



•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


•“Wet” and “Dry” Chip set-up

•Principle

•Combination of Biological and Electrical chips

Proposed Chip Concept

Circuitry

A

e-

e-

e-

Nano DNA Array


Thank You For Your Time

DNA Chip Team

Raphael Anstey

Mattheiu Chardon

Travis Harper

Questions?

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

Documents

dna slide

dna chip teamdecember

human dna

dna structure

dna sequence

dna principles

region of dna

dna strand