DNA microarray

DNA microarray:
A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.

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DNA microarray
Isfahan University of Medical Science, School of Pharmacy
Department of Clinical Biochemistry

2. DNA
MICROARRAY
(An overview)
By:
A.N. Emami Razavi

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DNA microarray
Outlines
Introduc tion
Applic a tions
Type s of D NA mic roa rra y
Mic roa rre y proc e s s
Mic roa rra y a na lys is

4. Introduction
DNA microarray

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DNA microarray
What is a Microarray?
Microarray” has become a general term, there are
many types now
DNA microarrays
Protein microarrays
Transfection microarrays
Antibody microarray
Tissue microarray
Chemical compound microarray
…
We’ll be discussing DNA microarrays

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DNA microarray
A DNA microarray (also commonly known as gene or
genome chip, DNA chip, or gene array) is a collection of
microscopic DNA spots, commonly representing single genes,
arrayed on a solid surface by covalent attachment to a
chemical matrix. DNA arrays are different from other types of
microarray only in that they either measure DNA or use DNA
as part of its detection system. Qualitative or quantitative
measurements with DNA microarrays utilize the selective
nature of DNA-DNA or DNA-RNA hybridization under high-
stringency conditions and fluorophore-based detection. DNA
arrays are commonly used for expression profiling, i.e.,
monitoring expression levels of thousands of genes
simultaneously.

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DNA microarray
The affixed DNA segments are known as probes
(although some sources will use different
nomenclature such as reporters), thousands of which
can be placed in known locations on a single DNA
microarray. Microarray technology evolved from
Southern blotting, whereby fragmented DNA is
attached to a substrate and then probed with a known
gene or fragment.

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DNA microarray
DNA microarrays can be used to detect DNA (e.g., in
comparative genomic hybridization); it also permits
detection of RNA (most commonly as cDNA after
reverse transcription) that may or may not be
translated into proteins, which is referred to as
"expression analysis" or expression profiling.

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DNA microarray
Since there can be tens of thousands of distinct
probes on an array, each microarray experiment can
potentially accomplish the equivalent number of
genetic tests in parallel. Arrays have therefore
dramatically accelerated many types of
investigations. The use of a collection of distinct
DNAs in arrays for expression profiling was first
described in 1987, and the arrayed DNAs were used
to identify genes whose expression is modulated by
interferon.

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DNA microarray
These early gene arrays were made by spotting
cDNAs onto filter paper with a pin-spotting device.
The use of miniaturized microarrays for gene
expression profiling was first reported in 1995, and a
complete eukaryotic genome (Saccharomyces
cerevisiae) on a microarray was published in 1997.

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Microarrays are Popular
PubMed search "microarray"= 23,559 papers (february 17,2008)
 // // = 28,027 papers (march,9,2008)
2005 = 4406
2004 = 3509
2003 = 2421
2002 = 1557
2001 = 834
2000 = 294
294
834
1557
2421
3509
4406
294
834
1557
2421
3509
4406
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12. Applications
DNA microarray

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Gene expression profiling
In different cells/tissues
During the course of development
Under different environmental or chemical stimuli
In disease state versus healthy
Molecular diagnosis:
Molecular classification of disease
Drug development
Identification of new targets
Pharmacogenomics
Individualized medicine

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DNA microarray
Comparative genomic hybridization
Assessing genome content in different cells or closely related
organisms.

SNP detection arrays
Identifying single nucleotide polymorphism among alleles within or
between populations.
Chromatin immunoprecipitation (ChIP) studies
 Determining protein binding site occupancy throughout the genome,
employing ChIP-on-chip technology.

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DNA microarray
Areas being studied with microarrays
Differential gene expression between two (or more) sample types
Similar gene expression across treatments
Tumor sub-class identification using gene expression profiles
Classification of malignancies into known classes
Identification of “marker” genes that characterize different tumor classes
Identification of genes associated with clinical outcomes (e.g. survival)

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DNA microarray
mRNA levels compared in many different contexts
Different tissues, same organism (brain v. liver)

Same tissue, same organism (tumor v. non-tumor)

Same tissue, different organisms (wt v. mutant)
Time course experiments (development)

17. Types of
DNA
Microarray
DNA microarray

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DNA microarray
Types of Microarrays
Spotted DNA arrays (“cDNA arrays”)
Developed by Pat Brown (Stanford)
PCR products (or long oligos) from known genes (~100 nt) spotted on
glass, plastic, or nylon support
Customizable and off the shelf
Gene Chips
Oligonucleotide arrays (Affymetrix)
Large number of 20-25mers/gene
Enabled by photolithography from the computer industry
Off the shelf
Ink-jet microarrays (Agilent)
25-60mers “printed” directly on glass
Four cartridges: A, C, G, and T
Flexible, rapid, but expensive

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DNA microarray
Spotted DNA arrays
In spotted microarrays, the probes are oligonucleotides, cDNA
or small fragments of PCR products that correspond to
mRNAs. There probes are synthesized prior to deposition on
the array surface and are then "spotted" onto glass. A common
approach utilizes an array of fine pins or needles controlled by
a robotic arm that is dipped into wells containing DNA probes
and then depositing each probe at designated locations on the
array surface. The resulting "grid" of probes represents the
nucleic acid profiles of the prepared probes and is ready to
receive cDNA derived from experimental or clinical samples.

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DNA microarray
Building a cDNA chip
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
PCR amplification of
target DNA
(cDNA or portion of
genomic DNA)
Consolidate
into plates
Spot as microarray
on glass slides

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DNA microarray
Make Chip
Glass slide
Robot spotter
Microarray “spotters” are high-precision robots
with metal pins that dip into DNA solution &
tap down on glass slide (pins work like a
fountain pen)

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DNA microarray
This technique is used by research scientists around the world
to produce "in-house" printed microarrays from their own labs.
These arrays may be easily customized for each experiment,
because researchers can choose the probes and printing
locations on the arrays, synthesize the probes in their own lab
(or collaborating facility), and spot the arrays. They can then
generate their own labeled samples for hybridization,
hybridize the samples to the array, and finally scan the arrays
with their own equipment. This provides a relatively low-cost
microarray that is customized for each study, and avoids the
costs of purchasing often more expensive commercial arrays
that may represent vast numbers of genes that are not of
interest to the investigator.

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DNA microarray
Oligonucleotide arrays
In oligonucleotide microarrays, the probes are short sequences
designed to match parts of the sequence of known or predicted
open reading frames. Although oligonucleotide probes are
often used in "spotted" microarrays, the term "oligonucleotide
array" most often refers to a specific technique of
manufacturing. Oligonucleotide arrays are produced by
printing short oligonucleotide sequences designed to represent
a single gene by synthesizing this sequence directly onto the
array surface instead of depositing intact sequences.

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DNA microarray
Sequences may be longer (60-mer probes such as the Agilent
design) or shorter (25-mer probes produced by Affymetrix)
depending on the desired purpose; longer probes are more
specific to individual target genes, shorter probes may be
spotted in higher density across the array and are cheaper to
manufacture.
One technique used to produce oligonucleotide arrays include
photolithographic synthesis (Agilent and Affymetrix) on a
silica substrate where light and light-sensitive masking agents
are used to "build" a sequence one nucleotide at a time across
the entire array.

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DNA microarray

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DNA microarray
Spotted Vs. Oligonucleotide array
Spotted Arrays
Relative cheap to make (~$10
slide)
Flexible - spot anything you
want
Cheap so can repeat
experiments many times
Highly variable spot
deposition
Usually have to make your
own
Affy Gene Chips
Expensive ($500 or more)
Limited types avail, no chance
of specialized chips
Fewer repeated experiments
usually
More uniform DNA feaures
Can buy off the shelf

27. DNA Microarray
Process
DNA microarray

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DNA microarray
Overview of the process
Questio
n
Sample
Preparatio
n
Data
Analysis
Microarray

Hybridizatio
n
Microarra
y
Detection

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DNA microarray
Gene Expression Patterns
Genes are expressed when they are copied into mRNA or RNA
(transcription)
Differential gene expression: which genes are expressed in which cells or
tissues at a given point in time or in the life of the organism.
Total RNA can be isolated from cells or tissues under different
experimental conditions and the relative amounts of transcribed RNA can
be measured
The change in expression pattern in response to an experimental condition,
environmental change, drug treatment, etc. sheds light into the dynamic
functioning of a cell

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DNA microarray

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DNA microarray

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DNA microarray

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DNA microarray

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DNA microarray
Experiment process
1.Collect tissue
2.Isolate RNA
3.Isolate mRNA
4.Make labeled DNA copy
5.Apply DNA
6.Scan microarray
7.Analyze data

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DNA microarray
Equipments

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DNA microarray
Collect tissue

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DNA microarray
Isolate RNA

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DNA microarray
Isolate mRNA

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DNA microarray
Make labeled DNA copy

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DNA microarray
Apply DNA

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DNA microarray
Scan microarray

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DNA microarray
Analyze data

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DNA microarray
Quantification
Cy5 at 635 Cy3 at 532
Overlay images

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DNA microarray
Expression profiling with DNA microarrays
Data acquistion
HYBRIDIZATION
Spot #1
Spot #3
Spot #2
Spot #4
Spot #6
Spot #5
Relative
intensity
Relative
intensity
cDNA “control”
Cy3 labeled
6
1
5
5
5
5
2
2
2
2
3
3
Output Output
cDNA “treament”
Cy5 labeled
1
6
6
6
5
5
5
5
3 6
6
4
5
3
6
6
6
6
6
6
1
1
5
5
5
5
5
5
5
2
2
2
2
3
3
4

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DNA microarray
Image Analysis & DataData Visualization
2001000050.005.64
480048001.00 0.00
90003000.03-4.91
Cy3Cy5
Cy5
Cy3
Cy5
Cy3
log
2
Genes
Experiments
8
4
2
fold
2
4
8
Underexpressed
Overexpressed

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DNA microarray
Sample Data

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DNA microarray
Data storage and retrieval
Filtering
Normalization
Analysis

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DNA microarray
Analysis of Microarray Data
Clustering
Idea: Groups of genes that share similar function have similar expression patterns
Hierarchical clustering
k-means
Bayesian approaches
Projection techniques
Principal Component Analysis
Independent Component Analysis
Classification
Idea: A cell can be in one of several states
(Diseased vs. Healthy, Cancer X vs. Cancer Y vs. Normal)
Can we train an algorithm to use the gene expression patterns to determine which state a
cell is in?
Support Vector Machines
Decision Trees
Neural Networks
K-Nearest Neighbors

51. Thank you
Questions ?

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DNA microarray
Two-color vs. one-color detection
Two-Color microarrays are typically hybridized with cDNA
prepared from two samples to be compared (e.g. diseased
tissue versus healthy tissue) and that are labeled with two
different fluorophores. Fluorescent dyes commonly used for
cDNA labelling include Cy3, which has a fluorescence
emission wavelength of 570 nm (corresponding to the green
part of the light spectrum), and Cy5 with a fluorescence
emission wavelength of 670 nm (corresponding to the red part
of the light spectrum). The two Cy-labelled cDNA samples are
mixed and hybridized to a single microarray that is then
scanned in a microarray scanner to visualize fluorescence of
the two fluorophores after excitation with a laser beam of a
defined wavelength. Relative intensities of each fluorophore
may then be used in ratio-based analysis to identify up-
regulated and down-regulated genes.