Download DNA Microarray kit

DNA MICROARRAYS
DNA Microarray
Micro Nano Tech Conference I - 2011
DNA Microarrays
DNA microarrays help us learn more about human diseases,
what causes them, how to identify them, and how to treat
them.
We now know more about complex diseases such as
diabetes, multiple sclerosis, heart disease, and cancer than
we have ever known before.
For some diseases researchers have been able to identify
specific genes that influence the risk of getting a disease.
They have found that most diseases that are affected by
one’s genes are influenced by many, many genes and not
just one or two.
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Review of DNA
Before jumping into the DNA
microarray, let’s review the
Deoxyribonucleic acid (DNA)
molecule, DNA transcription,
and DNA hybridization.
Please refer to the glossary at
the end of the Primary
Knowledge unit if you get
stuck on terminology.
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What is DNA?
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DNA is a long polymeric molecule
(found in most cells) that
functions in the chromosome as
the carrier of genetic information.
The genetic information is stored
in the linear sequences of the
base pairs (nucleotides):
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Adenine-Thymine (A-T or T-A)
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Guanine-Cytosine (G-C or C-G)
One DNA molecule may consists
of millions of nucleotides and
thus, millions of linear sequences
(genes).
A,T, G, and C are the nitrogenous bases. When paired,
they are called base pairs or nucleotides. Each base
pair is joined with a weak hydrogen bond.
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Your Genes
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An organism’s “genome” is its hereditary information
carried in DNA.
The Human Genome project (1990 through 2003)
mapped approximately 30,000 linear sequences or
human genes.
Every single cell in the human body contains the exact
same genes; however, some are “active” and other’s are
not.
DNA Microarrays identify specific genes as well as the
activity of genes. (More on that later.)
DNA microarrays use “copies” of one’s DNA.
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DNA Transcription
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DNA copies are made through DNA Transcription
followed by reverse transcription.
Transcription creates a messenger Ribonucleic acid
molecule (mRNA) from a DNA molecule.
The DNA sequences (genes) from one half of the DNA
molecule are “copied” into the mRNA.
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Reverse Transcription
Through reverse transcription the mRNA sequences are
transferred into a DNA copy or cDNA.
DNA microarrays require the
cDNA rather than the mRNA,
because mRNAs are
unstable, thus leading to
inaccurate and unreliable
results. cDNA are less likely
to degrade during the
hybridization process in a
microarray.
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DNA Hybridization
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DNA hybridization is when
a single-stranded DNA
molecule (ssDNA or the
cDNA) reanneals with
another ssDNA from
another source.
If the cDNA strands have
sequences that are
complementary to the
introduced ssDNA strands,
they can form a dsDNA
hybrid molecule with one
strand from each.
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DNA Hybridization Animation
Play QuickTime Animation
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DNA Microarrays and Hybridization
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DNA microarrays use gene sequencing, DNA
transcription and reverse transcription, and hybridization
to analyze and identify thousands of genes
simultaneously.
Each microarray consists of hundreds or thousands of
gene sequences (or ssDNA molecules) mounted on a
chip and used as “probes”.
Theses probes are called oligonucleotides or oligos.
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What is a DNA Microarray?
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The DNA microarray
relies on the
hybridization of cDNA
fragments to synthetic
ssDNA sequences
(specific A, C, G, T
combinations).
The synthetic
fragments (oligos) are
the probes.
The cDNA fragments
are the targets.
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DNA Microarrays
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A DNA microarray is a grid on a
substrate (e.g., glass, silicon).
Each position in the grid is an
“address” or “feature” as small as
200 nm square.
Each feature may contain
hundreds or thousands of identical
probes (oligos).
Each array may contain tens of
thousands of features.
Each feature is looking for a
specific gene or gene sequence.
Thousands of specific genes can
be identified simultaneously.
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DNA Microarrays
This graphic illustrates one feature of
a DNA microarray expanding from
many features (bottom grid) to a few
features (middle grid), to a single
feature (top grid) depicting a unique
DNA sequence (G-T-A-C-T-A…).
The coloration in this graphic is strictly
to illustrate different locations
(features) of ssDNA sequences
(oligos) in a DNA microarray.
DNA, and thus a DNA microarray is
actually colorless.
In the middle graphic,
how many gene
sequences are being
tested?
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DNA Microarrays “chips”
The image below shows a DNA microarray with tens of
thousands of features (left) and an exploded section of the array
(right). Each colored dot is one “feature” or “address”
containing hundreds/thousands of the same oligo probe that
have hybridized with cDNA from a sample. [Courtesy of Affymetrix]
We’ll explain the colors in a later slide.
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DNA Microarray Controls
Controls are used to ensure the test results by ensuring the
accuracy of the microarray’s fabrication and the preparation of
the samples (control and test samples).
Control sample – A set of cDNA from tissue for which the genes
are known.
Test sample – A set of cDNA from tissue that is to be analyzed.
Positive control – A feature that must show hybridization with
both the control sample and test sample.
Negative control – A feature that must show NO hybridization
with any cDNA from either sample.
Direct Comparison controls – Each feature of the array is a
comparison between the control sample and the test sample
(patient).
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How Does a DNA Microarray Work?
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[Courtesy of "The Science Creative Quarterly". scq.ubc.ca. Artist: Jiang Long}
DNA Microarrays use a
“control” and “target” or
experimental cell.
1. For each cell, mRNA is
extracted from the
“active” DNA in the cells.
2. cDNA is reversed
transcribed from the
mRNA.
3. Each cDNA is
fluorescently labeled
green (control) and red
(target).
4. The samples are
combined and washed
over the array.
5. Complementary genes
hybridization with the
synthetic probes on the
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array.
Interpretation of Microarray Results
[Image courtesy of NASA]
A flourescening DNA microarray showing
the results of DNA hybridization between
the probes and target DNAs. When
cDNA is prepared from a test sample
(red) and from a control sample (green),
and both hybridize with probes, the color
of the dot indicates the relative activity
level or the presence of that gene in one
or both samples.
Green dot shows activity or presence
in the control only
Red shows activity or presence in the
test tissue only
Yellow shows activity/presence in
both.
Black is activity/presence with neither
sample.
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Types of DNA Microarrays
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Gene expression microarrays detect
“expression levels” in a sample - when
mRNA copies to cDNA (which genes are
“active” or “inactive”). Gene expression
microarrays detect how cells and
organisms change and adapt to specific
stimuli such as changes in the
environment or one’s disease state.
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Direct detection microarrays are used to
identify specific genes that cause a
specific disease, and to screen for
mutations that are responsible for genetic
disorders when there are multiple gene
mutations that can possibly cause the
disorder.
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This image is the gene expression data
matrix of 70 prognostic markers genes
from tumors of 78 breast cancer patients
hybridized using a DNA microarray
referred to as the MammaPrint.
Each row represents a tumor tissue from a
patient and each column a gene.
[This image is public domain]
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DNA Microarray Animation
Play QuickTime Animation
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DNA Microarray Applications
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Identify specific genes that cause a specific disease
Identify gene mutations
Compare genes between different organisms
Create gene expression profiles (A map that shows gene
activity or the making of mRNA. Gene expression profiles detect
how cells and organism change and adapt to specific stimuli such
as change’s in environment or one’s disease state.)
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Study cancer causing genes
Customize drug therapies based on one’s personal
genes
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DNA Microarray Fabrication
So how on earth do we fabricate a DNA
microarray?
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GENECHIP MODEL
ACTIVITY
DNA Microarray Learning Module
Activity Description
In this activity you study how DNA (Deoxyribonucleic
acid) microarrays are fabricated using a
photolithography process developed by the
semiconductor manufacturing industry.
You then apply this knowledge to building a macro-size
DNA microarray model.
This activity should improve your understanding of how
DNA microarrays are made and how they identify
complementary single-stranded DNA (ssDNA) in a
sample.
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Activity Objectives and Outcomes
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Using the components provided in a SCME DNA Microarray kit, build
a macro-size DNA microarray with a three (3) nucleotide sequence.
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Outline and explain the fabrication steps for an oligonucleotide array
or GeneChip®.
At the end of this activity, you will be able to answer the following
questions:
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How are oligonucleotides used in a DNA microarray?
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How are synthetic oligonucleotides fabricated on a DNA microarray?
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How does a DNA microarray identify different target molecules
simultaneously?
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DNA Microarray Fabrication
One type of microarray fabrication technique uses a printer
similar to an ink-jet printhead (a micro-size device) to print the
addresses onto the microarray slide. However, the “ink” is an
oligonucleotide solution that deposits one nano-size nucleotide
at a time, creating the desired nucleotide sequences (or
oligonucleotides).
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DNA Microarray Fabrication
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The inkjet printing of a DNA microarray is very laborious due to all of
the oligonucleotide “inks” that must be prepared for each of the
addresses on the microarray.
These oligos are synthesized by chemical methods, one at a time,
or are prepared enzymatically by a reverse transcription of mRNA
isolated from cells to copy DNA or cDNA.
Alternatively, a photolithography method uses the photolithography
process borrowed from the semiconductor fabrication industry in
combination with chemical reactions to synthesize oligonucleotide
(oligo) probes on a silicon surface.
The oligos on these arrays are generally 20 to 25 nucleotides long
and each feature in the array itself may be as small as 50 nm
square – almost 2000 times smaller than the width of a strand of
hair!
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Microarray Fabrication - Photolithography
This method duplicates the photolithography process used in microfabrication
along chemical reactions between silicon and a nucleotide, and between
different nucleotides.
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A patterned mask and ultraviolet (UV)
light “expose” specific nano-size
features of the microarray. Each
feature is a location for several
nucleotides of the same sequence.
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The graphic illustrates the UV light,
mask, and substrate.
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Each square in the mask is a select
feature or address in the array.
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Inside each feature are several silicon
molecules that will “link” with the initial
nucleotides.
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This is the method that we will explore in the GeneChip®
Model Activity. This method was developed by Affymetrix
and is used to fabricate synthetic oligonucleotides on a
silicon substrate.
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Fabrication of a Oligonucleotide Array
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The light that travels through
these holes initiates the growth
of a nucleotide chain through a
process of deprotection and
addition of new bases into
specific chains / probes.
The chain begins with a
nucleotide linking to an
unprotected silicon molecule
on the surface of the substrate.
The mask is therefore the tool
that ultimately controls the
building of the oligo probes.
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Three Step Process
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Protect – Initially, a blocking compound is washed over
the entire silicon wafer. In subsequent steps, the
blocking compound is already attached to the
nucleotides in the Addition step.
Deprotect (Photolithography) – UV light through the
“holes” in the mask, removes the blocking compound,
“deprotecting” that area.
Addition – The substrate is washed with a solution of the
specific nucleotide being added at that step. The
nucleotides in the solution attach to the deprotected
areas on the wafer.
This cycle is repeated for each new nucleotide.
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“Genechip” Animations
 Let’s
take a look at a couple of animations.
GeneChip Animation on YouTube
DNA Microarray by Affymetrix
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Let’s to do it!
 Are
you ready?
 Let’s build a three nucleotide sequence DNA
microarray!
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GeneChip ® Model Activity
So let’s step through
activity materials and
fabrication process
together.
The board simulates an
array on a silicon
substrate. (Yours may look a little
different from the one in the picture.)
By the end of this activity,
each feature in the array
will have an oligo of 3
nucleotides.
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GeneChip ® Model Activity
In this activity you will fabricate
a 3 nucleotide oligo (nucleotide
sequence) using 12 masks.
 Each mask identifies the
locations for one of four
nucleotides (A, C, T, or G)
with a hole or opening.
 Colored beads are used for
each nucleotide. Refer to
the top of your substrate
(board) for the color of bead
vs. the specific nucleotide.
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GeneChip Model Activity (Fabrication)
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Set up your station with your substrate, beads (nucleotides), and
mask (cards with the pretty flowered holes).
Using mask 1, align it over the substrate using the 2 alignment
pegs at the top of the board.
Mask 1 is for the T nucleotides. Drop a T bead through each hole
and onto a nail. (You have just “exposed for” and “added” a T
nucleotide.
Remove the Mask 1. Align Mask 2 for the C nucleotide.
Add C to all of the deprotected areas.
Continue this process through all 12 masks.
On the array table in your activity, write the oligo for each feature.
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Interpretation of Microarray Results
A flourescening DNA microarray showing
the results of DNA hybridization between
the probe and target DNAs. . When
cDNA is prepared from a test sample
(red) and from a control sample (green),
and both are hybridized to the microarray,
the color of the dot indicates the relative
activity level of that gene. A green dot
shows activity in the control only, red
shows activity in the test tissue only, and
yellow shows activity in both. Black is
activity in neither sample.
[Image courtesy of NASA]
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GeneChip Model Activity (Interpretation)
Remember this graphic?
Ask your instructor for a sample
bag. In your bag are fluorescently
labeled nucleotides (red or green
sequin with 3 beads).
These are cDNA targets from a
control cell and test cell. Which is
which?
Match your targets with the oligo
probes on your array.
Which genes are detected in the
control and in the target?
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Summary
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DNA microarray fabrication requires the construction of
thousands or millions of oligonucleotides within the
features of an array.
One fabrication process uses the technology of the inkjet
printer while another process uses photolithography.
The photolithography process results in synthetic oligos
built one nucleotide at a time using specially designed
masks and UV light.
Each of these processes has the ability to construct large
microarrays capable of identifying thousands of different
genes simultaneously.
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Incorporating bioMEMS
How could you incorporate the information
from this workshop into your curriculum?
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THANK YOU FOR COMING!!!!
Please check the SCME website
periodically - scme-nm.org.
We are always uploading new materials
and scheduling new workshops.
Feel free to participant in our forum to
present ideas, results, and ask questions.
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