Principle and

Application of
DNA
Microarray
©Sources
1. Dr Ashish C Patel
2. Dr. Holly Dressman
3. Ruchi yadav
4. Mahendra Reddy

Genomics & Proteomics Biology Department

Faculty of Mathematics and Natural Sciences
Universitas Indonesia
INTRODUCTION
• A human organism
has over 250
different cell types
(e.g., muscle, skin,
bone, neuron),
most of which have
identical genomes,
yet they look
different and do
different jobs
INTRODUCTION
• It is believed that less
than 20% of the genes
are‘expressed’ (i.e.,
making RNA) in a
typical cell type
• Apparently the
differences in gene
expression is what
makes the cells
different
All of the cells within an individual conatin the
exact same genetic information

4
Genes can be turn “on” and “off”

5
Different sets of genes are turned “on” in different
types of cells

6
Gene Expression: The “Central Dogma”
 Microarrays:
Universal Biochemistry Platforms
Proteins
Peptides DNA

Lipids

Carbohydrates Small molecules

 What is a DNA Microarray?
• Biological Samples in 2D Arrays on
Membranes or Glass Slides

Cheung et al. 1999

What is a DNA Microarray?

• A DNA microarray (also

commonly known as DNA Chip
or Biochip) is a collection of
microscopic DNA spots
attached to a solid surface. A
snapshot that captures the
activity pattern of thousands
of genes at once.
What is a DNA Microarray?

• Each DNA spot contains

picomoles (10−12 moles) of a
specific DNA sequence, known as
probe (or oligos).

• 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.
What is a DNA Microarray?

• Fluorescently labeled target

sequences that bind to a probe
sequence generate a signal.
Historical background :
 Principle

• The core principle behind microarrays is hybridization.

At least two samples are hybridized to chip. Using this
technology the presence of genomic or cDNA sequence
in 1,00,000 or more sequences can be screened in a
single hybridization
 Principle

• Complementary nucleic acid sequences get pair

via hydrogen bonds between complementary
nucleotide base pairs.
• Samples are labeled using fluorescent dyes
• Washing off of non-specific bonding sequences .
 The Colours of a Microarray
• GREEN represents Control
DNA, where either DNA or
cDNA derived from normal
tissue is hybridized to the
target DNA.

• RED represents Sample DNA,

where either DNA or cDNA is
derived from diseased tissue
hybridized to the target DNA.
 The Colours of a Microarray

• YELLOW represents a
combination of Control and
Sample DNA, where both
hybridized equally to the target
DNA.

• BLACK represents areas where

neither the Control nor
Sample DNA hybridized to the
target DNA.
 The Colours of a Microarray

• Tables are difficult

to read
• Data is presented
with a color scale
 The Colours of a Microarray

• Coding scheme:
– Green = repressed
(less mRNA) gene in
experiment
– Red = induced (more
mRNA) gene in
experiment
– Black = no change (1:1
ratio)
 The Colours of a Microarray

• Or
– Green = control
condition (e.g.
aerobic)
– Red = experimental
condition (e.g.
anaerobic)
• We only use ratio
Microarray Experiment
Microarray Steps

• Experiment and Data Acquisition

– Sample preparation and labelling
– Hybridisation
– Washing
– Image acquisition

• Data normalization
• Data analysis
• Biological interpretation
Experiment and Data Acquisition

1. Sample
preparation

2.Purification
Experiment and Data Acquisition
3. Reverse Transcription

4. Labelling: Cyanine dyes Cy3

and Cy5 used predominant
label in microarray analysis
Experiment and Data Acquisition

5. Hibridization

6. Scanning

7. Normalizatio and
analysis
MICROARRAY TYPES

Two popular microarraying platforms:

Spotted Commercially available
microarrays microarray
MICROARRAY TYPES
Spotted Microarray
• 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.
MICROARRAY TYPES
Spotted Microarray
• 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.
MICROARRAY TYPES
Spotted Microarray
• 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.
Building 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
MICROARRAY TYPES
Oligonucleotide Microarray
• Gene chip (DNA chip, Affymetrix chip):
• In oligonucleotide microarrays, the probes
are short sequences designed to match
parts of the sequence of known or predicted
open reading frames. Oligonucleotide
(20~80-mer oligos) is synthesized either in
situ (on-chip)
MICROARRAY TYPES
Oligonucleotide Microarray
• 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.
Oligonucleotide Microarray
• 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.
Photolithographic masking method: used in the semiconductor
industry, in which attaching chemically modified linker groups,
which contain photochemically removable protective groups,
onto
the glass surface.
• Target synthesis proceeds in a step-wise fashion. In each step,
the unprotected areas are first activated with light which
removes the light sensitive protective groups.
• Exposure of the activated areas to a nucleoside solution
results in chemical attachment of the nucleoside to the
activated positions. This process is then repeated by using a
different mask and a new nucleotide until all nucleotides
have been added to the oligo
 Photolithography Method

The mask only allows light to pass to specific features on the chip
Photolithography

Photolithography: UV light is passed through a

lithographic mask that acts as a filter to either transmit or
block the light from the chemically protected microarray
surface (wafer). The sequential application of specific
lithographic masks determines the order of sequence
synthesis on the wafer surface. (Bottom) Chemical
synthesis cycle. UV light removes the protecting groups
(squares) from the array surface, allowing the addition of
a single protected nucleotide as it is washed over the
microarray. Sequential rounds of light deprotection,
changes in the filtering patterns of the masks, and single
nucleotide additions form microarray features with
specific 25-bp probes
Photolithography
 Affymetrix GeneChips

▪The black features represent no

intensity (no RNA hybridized to the
respective probe in the feature).
 Affymetrix GeneChips

The intensity level from lowest to highest by color is: Dark

blue -> Blue -
> Light Blue -> Green -> Yellow -> Orange -> Red - > White.

▪More intensity means more RNA bound to a specific

feature, which basically means the gene was
expressed at a higher level.
Affymetrix GeneChip experiment
Affymetrix GeneChip experiment
 Spotted Vs. Oligonucleotide Array
Spotted Arrays Affy Gene Chips
❑ Relative cheap to make
(~$10 slide) ❑ Expensive ($500 or more)
❑ Flexible - spot anything
you ❑ Limited types avail, no
want chance of specialized
❑ Cheap so can repeat
experiments many chips
times ❑ Fewer repeated
❑ Highly variable
spot deposition experiments usually
❑ Usually have to make ❑ More uniform DNA
your
own features
❑ Can buy off the shelf
 Fabrication of Microarray
• Printed array
– Robotic Spotting: Contact printing via a variety of pins
– Ink-jet printing: Non contact printing
• In-Situ Oligonucleotide synthesis
– Photolithography
– Ink-jet: On Chip Synthesis
• High Density Bead arrays
– Sequence tagged beads are randomly assorted onto
fiberoptic bundles or silicone slides
• Electronic microarray
– Microelectrode arrays, electrophoretic transport to load
capture probes, hydrogel permeation layers
Robotic Spotting
Probes are PCR amplified (ooligonucleotides are
synthesized) and subsequently spotted onto a
glass slide
Agilent oligonucleotide microarray by InkJet
technology
(A) Noncontact inkjet printing
technology delivers a small
and accurate volume
(picoliters) of nucleotides to
the first layer on the
microarray surface.
(B) Repeated rounds of base-
by-base printing extend the
length of specific
oligonucleotide probes.
(C) Close-up of growing
oligonucleotide chain with a
base being added.
(D) The final product is a 60-mer
in situ-synthesized probe as
a feature on a microarray
containing thousands of
specifically synthesized
probes.
High density bead array

The SAM contains 96

1.4-mm fiber-optic bundles (bottom left). Each bundle is
an individual array consisting of 50,000 5-μm fiber-optic
strands, each of which is chemically etched to create a
microwell for a single bead (top left). The Sentrix Bead
Chips can assay 1 to 16 samples at a time on a silicon
slide (bottom right) that has been processed to provide
microwells for individual beads (top right). Both Bead
Array platforms rely on 3-μm silica beads that randomly
self- assemble (center).
High density bead array
Electronic Microarray
A key feature unique to electronic microarrays is
that electronic hybridization occurs within 1–2
min
•Image of 100-site microarray. Platinum working electrodes in a 10x10
array are connected by platinum wires to contact pads located on the
periphery of the chip.
•Surrounding the 10 x10 array are 20 platinum counter electrodes.
•The 80-µm diameter platinum working electrodes are coated with a
hydrogel permeation layer.
•The hydrogel contains covalently bound streptavidin for binding biotin-
labeled oligonucleotides
(A) A positive electric current is applied totest sites, facilitating the active movement
and concentration of negatively charged DNA probes to the activated locations.
(B) Once the first probe is bound to its targeted location(s) by streptavidin-biotin
bonds, the test site(s) can be deactivated, and current can be applied to a
different test site. This process is repeated until all the probes are arrayed.
Electronic Microarray
Applications of Microarrays
Gene Target Discovery
-Diseased vs normal
cell comparison
suggests sets of genes
having key roles.
-Over/underexpressed
genes in the diseased
cells can suggest drug
targets
Applications of Microarrays

Pharmacology and
Toxicology
-Highly sensitive
indicator of a
drug’s activity
(pharmacology)
and toxicity
(toxicology) in
cell culture or
test animals.
-Screen or optimize drug
candidates prior to costly
clinical trials.
Applications of Microarrays

Diagnostics
-Potential to diagnose
clinical conditions by
detecting gene
expression patterns
associated with
disease states in
either biopsy
samples or
peripheral blood
cells.
 Applications of Microarrays
Gene expressionanalysis
• The process of
measuring gene
expression via
cDNA is called
expression
analysis.
• Not all the genes
in the human
genome are active
at all times.
 Applications of Microarrays
Gene expressionanalysis
• Used to detect
DNA , or detect
RNA that
may or may not
be translated
into proteins.
• Thousand genes
are simultaneously
assessed.
 Applications of Microarrays
Gene expressionanalysis
• Study the
effects of
certain
treatments,
diseases, and
development
al stages on
gene
expression.
Exp: Gene expression and obesity

*Measuring levels of
gene expression

*Creating diagnostic
tests to predict
whether a patient
has a genetic
predisposition to
obesity

*Designing Drugs
 Microarrays promise to speed up drug discovery,
provide safer and more personalised medicines and
eradicate disease in an argument that goes
something like this. Aberrant gene function
(mediated by proteins) causes every human disease
and the sequence of every disease-causing gene is
now known. Millions of small molecules made
available through the combined sources of natural
products, organic synthesis and combinatorial
chemistry, represent a yet-to-be-discovered drug
against every human protein.
In some cases, genes, genes products and derivatives thereof can be
used to ameliorate disease. Microarray assays allow the examination
of DNA, RNA, proteins, small molecules and tissues in a massively
parallel format, allowing partnering between every human gene
(protein) and a specific therapeutic agent (Figure 6).
 TROUBLESHOOTING MICROARRAY EXPERIMENTS
No hybridization signal
Possible Causes Remedy
Target concentration too low. Determine target concentration before slide spotting.
Targets not clean enough. Remove PCR components from targets before slide
spotting.
Poor retention of targets on slide. Prepare new microarray slides. Check that spotting
buffer and protocol are compatible with slide type.
Failed labelling reaction. Always check the success of labelling reaction before
using it in hybridization.
Loss of probe during purification Check success of probe purification before use.
Poor hybridization. Check that hybridization buffer and protocol are
compatible with slide type.
Target genes not expressed in Use housekeeping genes and positive controls to
examined tissue. ascertain proper functioning of the system.
High background, weak specific signals.

Poor labelling reaction. Check success of labelling reaction specific signals.

before hybridization.
 TROUBLESHOOTING MICROARRAY EXPERIMENTS
Low or undetectable Cy3 and/or Cy5 signal
Possible Causes Remedy
Loss of probe in purification. Check performance of purification. Do not
purify Cy3 and Cy5 probes together.
RNA contaminated by DNA. Use DNAse I to remove DNA
Unequal amount of Cy3 and Cy5. Use equal amounts of probes
in hybridization.
Unbalanced Cy3 and/or Cy5 signal
Too much CyDye nucleotide in Use less CyDye nucleotide.
labelling reaction.
Bubble effect on slide
Air has been trapped under coverslip Remove air bubbles from hybridization
Microarray software
Microarray
Databases
and Tools

62
 Microarray tools
• NetAffix Analysis center from affymetrix
▪ Array content information
▪ Probe sequences
▪ Gene annotations
• Xcluster- tool for cluster analysis
• GENECLUSTER
• TIGR Microarray
▪ MADAM-Microarray data manager
▪ SPOTFINDER-image processing tool
▪ MIDAS-Microarray data analysis system
▪ MEV-MultiExperiment Viewer
ARRAY EXPRESS (EBI)
GEO(NCBI)
 Thank You

Genomics & Proteomics Biology Department

Faculty of Mathematics and Natural Sciences
Universitas Indonesia