DNA
MICROARRAY
Presented by,
Prof. P. D. Shelke
Department of Plant biotech,
K.K. Wagh college of agril biotechnology
Department of Plant biotech
DNA microarray
O u t l in e s
❑ In t r o d u c t io n
❑ A p p l ic a t io n s
❑ Typesof DNA microarray
❑ M ic r o a r r e y p r o c e s s
❑ M ic r o a r r a y a n a ly s i s
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of Plant
June 26,biotech
2012 3 Total slides : 51
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 may notbe
or
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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Plant biotech
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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DNA microarray
Microarrays are Popular
❑
PubMed search "microarray"= 23,559 papers (february 17,2008)
❑
// // = 28,027 papers (march,9,2008)
5000
4500 4406
❑ 2005 = 4406 4000
3500 3509
❑ 2004 = 3509 3000
❑ 2003 = 2421 2500 2421
2000
❑ 2002 = 1557 1500 1557
❑ 2001 = 834 1000
834
500
❑ 2000 = 294 294
0
2000 2001 2002 2003 2004 2005
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Plant biotech
Applications
DNA microarray
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DNA microarray
❑ 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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Plant biotech
DNA microarray
❑ Comparative genomic hybridization
❑ Assessing genome content in different cells or closely
organisms. related
❑ 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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Plant biotech
DNA microarray
Areas being studied with
microarrays
❑ Differential gene expression between two (or more) sample types
❑ Similar gene expression across treatments
❑ Identification of “marker” genes that characterize different tumor
classes
❑ Identification of genes associated with significant outcomes
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DNA microarray
mRNA levels compared in many different
contexts
❑ Different tissues, same organism
❑ Same tissue, same organism
❑ Same tissue, different organisms
❑ Time course experiments
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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
PCR amplification of Consolidate
target DNA into plates
(cDNA or portion of
genomic DNA)
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
Spot as microarray
on glass slides
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DNA microarray
Make Chip
Microarray “spotters” are high-precision robots
with metal pins that dip into DNA solution & Robot spotter
tap down on glass slide (pins work like a
fountain pen)
Glass slide
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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
❑ 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
▪Affy Gene Chips Spotted Arrays
❑ ❑
▪ Expensive ($500 or more) Relative cheap to make (~$10
❑
slide)
▪❑ Limited types avail, no chance of
specialized chips Flexible - spot anything you
❑
▪❑ Fewer repeated experiments want
usually ❑
Cheap so can repeat
❑
▪ More uniform DNA feaures experiments many times
❑
Highly variable spot deposition
❑
▪ Can buy off the shelf Usually have to make
your own
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DNA microarray
DNA MICROARAY
PROCESS
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DNA microarray
Overview of the process
Questio
n
Data Sample
Analysis Preparatio
n
Microarra
y Microarray
Detection
June 26, 2012 28 Hybridizatio
Department of Plant biotech Total slides : 51
DNA microarray
Gene Expression Patterns
❑ Genes are expressed when theyare copied into or
mRNA (transcription) RNA
❑ 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
Department of Plant biotech
DNA microarray
Department of Plant biotech
DNA microarray
Department of Plant biotech
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
Quantifica
tion Cy5 at 635 Cy3 at 532
Overlay images
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DNA microarray
Expression profiling with DNA
microarrays
cDNA “control” Data acquistion cDNA “treament”
Cy3 labeled Cy5 labeled
1 1 6
2 3 6
2 5
5 5 5
3 2 5
3 4 5
6 2
5 6 6
6 5
Relative Relative
Output HYBRIDIZATION Output
intensity intensity
1
Spot #1 Spot #4
1 4
2
2 5 5
Spot #2 5 5 Spot #5
2 2 5
5
5
3 56
3 Spot #3 6 Spot #6
3 6
6
6 6 of Plant biotech
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DNA microarray
Image Analysis & Data Visualization
Cy5 Cy5
log2 Cy3
Cy3 Cy5 Cy3
200 10000 50.00
5.64 0.0
4800 4800 1.00 0
9000 300 0.03 -4.91
Experiments
Underexpressed 8
4
2
Genes
2
4
Overexpressed 8
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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
❑ 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
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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).
52
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Total slides : 51
Contd...
❑ 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.
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Thank you
Questions ?
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