This ppt. will give valuable information about DNA microarray..

1. 1
DNA
MICROAARRAY
(DNA CHIP)
By: Dr Ashish C Patel
Assistant Professor
Vet College, AAU, Anand

2. 2
A DNA microarray is composed of pieces of DNA
ranging from 20-5000 base pairs concentrated into specific areas
on a solid support such as a glass or silicon. Each spot of DNA,
called a probe, represents a single gene. Each spot may contain a
few million copies of identical probe molecules.
There are several synonyms of DNA microarrays such as
DNA chips, gene chips, DNA arrays, gene arrays and biochips.
Microarray technology evolved from Southern blotting,
where fragmented DNA is attached to a substrate and then probed
with a known DNA sequence.
The use of microarrays for gene expression profiling was
first reported in 1995, and a complete eukaryotic genome
(Saccharomyces cerevisiae) on a microarray was published in
1997.

3. Overview
Array Production
Probe generation &
Robotic spotting
OnChip Probe synthesis
Sample Preparation
Cells/Tissues-
DNA/RNA/Protein/
other biomolecules-labelling
Hybridization
Stringency Washes
Image Processing
Scanning, Quantification,
Normalization
Data Analysis & Interpretation

4. 4
Principle
The principle of DNA microarrays lies on the
hybridization between the nucleotide. Using this
technology the presence of genomic or cDNA sequence
in 1,00,000 or more sequences can be screened in a
single hybridization.
The property of complementary nucleic acid
sequences is to specifically pair with each other by
forming hydrogen bonds between complementary
nucleotide base pairs.

5. 5
Design of a
DNA
Microarray
System

6. 6
1. Sample
preparation
2. Purification

7. 7
3. Reverse Transcription
4. Labelling: Cyanine dyes Cy3
and Cy5 used predominant label in
microarray analysis

8. 8
5.Hibridization
6. Scanning
7.Normalization
and analysis

9. Types of microarray
• DNA Microarray
– cDNA microarray
– Oligonucleotide arrays
• Protein microarray
– Analytical
– Functional
– Reverse phase
• Chemical compound arrays
– collection of organic chemical compounds spotted on a solid surface
• Carbohydrate arrays
– various oligosaccharides and/or polysaccharides immobilized on a solid
support in a spatially defined arrangement
• Cellular Microarrays
– spotted with varying materials, such as antibodies, proteins, or lipids,
which can interact with the cells, leading to their capture on specific
spots

10. DNA microarray
• Thousands of small “spots” or “features,”
• Millions of strands of the same sequence within the spot
covalently attached to the microarray surface
• The amount of DNA present in the spot correlates with the
overall binding capacity of the spot
• The larger the binding capacity, the greater the amount of
fluorescence signal that can be detected
• Binding capacity of the spot represents the detection range of
the microarray assay
• Each spot must contain sufficient binding sites to adequately
represent differences in expression levels

11. • Spot is created by placing DNA “probes” on the functionalized
surface
• Probes come in two distinct forms: oligonucleotide and PCR
probes (“cDNA probes”)
• An oligonucleotide probe is a single-stranded DNA that can
range in size typically from 20 to 80 nucleotides in length and
is synthesized using standard phosphoramidite chemistry.
• The cDNA probe is essentially a PCR product (of almost any
length) that is attached to the microarray surface using a
specific attachment chemistry or simply ultraviolet cross-
linking

12. • The decision to utilize oligo-probes or PCR probes depends
upon the amount of genomic information known about the
organism or cell system under investigation
• It is nearly impossible to design oligo-probes for organisms
where no genomic data are available
• Gene expression studies in “emerging” organisms (i.e., those
with little genomic data available) often involve PCR products
derived from a cDNA library
• Advantages of oligo-probes are -multiple oligo-probes can be
designed to a single gene, targeting oligo-probe designs to
specific exons or exon boundaries to essentially avoid potential
cross hybridization with non-target genes

13. • PCR probes can be attached to amine-reactive surfaces using
the amine groups native to DNA, or by adding a 5’ amino-
modifier to the PCR primer
• Once the microarrays have been spotted, a subsequent step is
to quench the reactivity of the remaining amino-reactive
surface on the microarray (i.e., the spaces between the spots)
• Spotting PCR probes on a poly-lysine microarray surface is
carried out in a similar manner, but steps are taken to link the
double-stranded PCR product to the surface
• This attachment method involves high heat (baking) and/or
ultraviolet cross-linking

14. 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

15. MANUFACTURING OF MICROARRAY SLIDES
• Microarray analysis is invariably performed on a glass slide,
which enables to perform hybridization assays with
fluorescently labelled samples.
• Microarray manufacture requires three distinct components:
• 1. Production method
• 2. Microarray slide
• 3. Target genetic content
15

16. 1. Production method
Oligo synthesis
• Two parallel approaches: a) Nucleic acid targets can either be
synthesized directly onto the microarray slide 2) purified targets
can be deposited onto a solid surface that is capable of binding
nucleic acids.
A) 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. 16

17. Affymetrix Oligonucleotide Microarray 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

18. Roche Nimblegen Oligonucleotide Microarray photolitography
Utilizes a digital micromirror
device (DMD) to create virtual
masks. The DMD forms the
pattern of UV light needed to
direct the specific nucleic acid
addition during photo-mediated
synthesis. UV light removes the
photolabile protecting group
(circles) from the microarray
surface, allowing the addition of
a single protected nucleotide to
the growing oligonucleotide
chain. The cycling of DMD
filtering, light deprotection, and
nucleotide addition creates
oligonucleotide features 60 to
100 bp in length on the
NimbleGen microarray

19. B) Deposition methods: purified nucleic acids are attached to a
modified glass slide. Typically, small volumes of nucleic acid
solution nanoliters or picoliters are transferred onto the glass slide.
• Deposition methods are equally suitable for preparing microarrays
containing oligonucleotides, cDNA sequences.
• The deposition chemistry involves a chemical reaction between
molecular groups on the glass surface and the oligo, resulting in
the formation of covalent bonds that bind the oligonucleotide onto
the array.
Array Spotter
• Array Spotter is a new contact deposition microarray spotter.
• Stainless steel capillary pens that conserve sample and uniformly
deposit picoliter volumes of target.
• From a single sample uptake of less than 200 nl, up to 150 spots
can be spotted in duplicate, across each of slide.
19

20. Robotic Spotting
Probes are PCR amplified (or
oligonucleotides are synthesized) and
subsequently spotted onto a glass slide

21. 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.

22. 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).

23. Electronic Microarray
(A) A positive electric current is applied to test
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.
• 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 key feature unique to electronic microarrays is that
electronic hybridization occurs within 1–2 min

24. Microarray slides
• Most commonly used support for microarrays are standard glass
microscope slides that offer flat and rigid support with low
intrinsic background fluorescence.
• Nucleic acids will not attach efficiently to an untreated glass slide.
The treatment not only enable the binding of targets.
• The uniformity and thickness of the surface coating on the slide is
critical for good quality microarray results.
• Variation in slide coating can contribute to the variation in
microarray signals and decrease the resolution of a microarray
experiment. Uneven slide coating can also lead to poor
attachment of deposited nucleic acid, which may come loose
during microarray hybridization.
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25. • Commonly used slide surface modifications include the
introduction of aldehyde, amino, or poly-lysine groups onto the
slide surface.
• Treated slides give highly consistent and reproducible data with
high signal to noise values, and they are most favorable for use in
microarray experiments.
Aldehyde slides
• To minimize fluorescent background.
• Aromatic amines on the G, C, and A bases of naturally occurring
DNA can also react with aldehyde groups.
Amine slides
• Amine groups can be introduced onto microarray slides by treating
cleaned glass with aminosilane .
• Vapor treatment of slides gives generally better results than
deposition by a dipping method.
25

26. Reflective slides
• A large proportion of the fluorescent light emitted from the
hybridized probe is scattered in all directions when using regular
glass arrays.
• The introduction of a reflective surface below the spotting
surface enables a significant amount of this scattered output to
be directed towards the detector, hence increasing the amount of
signal detected by the system.
• These reflective slides are constructed by adding a layer of
aluminium above the glass surface.
26

27. Target nucleic acids
• The third critical component in microarray manufacturing is the
target nucleic acid.
• Microarray targets must be available in high enough
concentration to allow a sufficient number of molecules to be
deposited onto the slide.
• The purity of target solutions is important for both the efficient
attachment of nucleic acids to the slide surface and the
availability of the immobilized targets for hybridization.
• PCR-amplified targets must be purified to remove dNTPs,
primers, DNA polymerase, buffer salts, and detergents.
• The targets, once attached to the microarray surface, are only
available for hybridization when they are present in a denatured,
single-stranded form.
• This can be achieved by spotting the targets under denaturing
conditions, with in high salt solutions, or in denaturing solvents
such as DMSO. 27

28. 28
Table 1. Steps in the design and implementation of a DNA
microarray
1) Probe
(cDNA/oligo
with known
identity)
2) Chip
fabrication
(Putting
probes on the
chip)
3) Target
(fluorecentl
y labeled
sample)
4) Assay 5) Readout
6)
Informatics
Small
oligonucleotides
,
cDNAs,
Photolithogra--
phy, pipette,
piezoelectric.
RNA,
(mRNA)
cDNA.
Hybridiza
tion.
Fluorescence,
probeless
(conductance,
MS,
electrophoresis
).
Robotics
control,
Image
processing,
DBMS,bioi
nformatics.

29. Sample Preparation for gene expression profiling by DNA
microarray
• First step in sample preparation for gene expression profiling
is RNA isolation from the biological sample
• The mRNA is converted to cDNA using reverse transcription
with fluorescently labelled nucleotides
• These fluorescence-labeled cDNAs represent the mRNAs in
the original sample and are hybridized to the microarray
• The two fluorescent dyes typically utilized in fluorescence
labeling are cyanine-3 (Cy-3) and cyanine-5 (Cy-5), which are
green- and red-colored dyes respectively

30. • Each microarray experiment involves two reverse transcription
reactions (e.g., control and drug-treated)
• The “control” (e.g., untreated) mRNA sample is added to a reverse
transcription reaction that includes a dye-conjugated nucleotide
(green)
• Whereas the “test” (e.g., drug-treated) sample is added to a reverse
transcription reaction that includes a different dye-conjugated
nucleotide (red)
• The cDNAs derived from the two reactions are mixed prior to
microarray hybridization, creating a “two-color” sample
• if the test sample (i.e., drug-treated sample) causes GENE X to
increase the mRNA expression levels, then the GENE X spot will
appear more red than green (after color channel normalization)
• If the green fluorescence (control sample) from the GENE X spot is
measured at 10,000 relative fluorescence units (RFUs) and the red
fluorescence (test sample) at 40,000 RFUs, then the test sample
contains a fourfold increase in GENE X expression (i.e., a 400%
increase over the control)

31. DNA microarray Hybridization
• The hybridization method(s) are aimed at placing the fluoro-
cDNA on the two-dimensional surface utilizing a stringency
conditions to facilitate sequence specific binding
• “Stringency” is a term used to describe the molecular
(thermodynamic) energy required for binding two
complementary, single stranded DNA molecules, which is
dependent largely on temperature, salt concentrations, and pH
• High stringency conditions involve high temperatures and/or
low salt concentrations, and DNA hybridizations proceed
slowly but in a sequence specific manner
• low stringency conditions involves cooler temperatures and/or
high salt concentrations, and DNA can form double-stranded
complexes even if their sequences are not complementary (i.e.,
nonspecific binding)

32. • Hybridization involves placing the fluoro-cDNA in a specific
buffer, and sandwiching a sample volume (50–500 mL) between
the DNA microarray and a cover slip or blank glass slide
• This assembly is then placed in a chamber where temperature,
and sometimes humidity, is controlled
• Typically, the hybridization needs more than 16–19 h (i.e.,
overnight) to allow sufficient time for the probes to bind to the
fluoro-cDNAs in a sequence-specific manner
• Once the incubation is complete, care should be taken while
removing the excess sample through a series of buffer washes
where stringency is controlled
• Finally the microarrays (slides) are dried using centrifugation or
airflow
• The microarrays are now ready for scanning (i.e., fluorescence
detection)

33. DNA Microarray Image processing
• Microarrays are placed in a microarray scanning instrument
• The spots will appear in varying colors from red to green to
yellow (yellow is a mixture of red and green fluorescence)
• If the control sample was labeled green and the drug treated
sample was labeled red, then a spot appearing red would
indicate that the gene (mRNA)expression increased during
drug treatment
• Spots lacking any color (fluorescence) indicate that the gene
(mRNA) was not expressed in the sample

34. • Once the microarray image has been derived using the scanner
(typically this is actually two images representing the red and
green images, and the scanner software displays an “overlay”
of these images), raw data analysis is needed to
– associate each spot with the gene (mRNA) that it is
detecting; and
– normalize the red and green channels to correct for any
differences in initial RNA concentrations, labeling reaction
efficiencies, and differences in the capabilities of each
channel (red and green) within the scanner itself

35. • Preprocessing of oligo arrays generally involves three steps:
background correction, normalization, and summarization
• Normalization in microarray experiments is carried out based
on the assumption that only a small proportion of genes will be
differentially expressed among the thousands of genes present
in the array and/or that there is symmetry in the up- and down-
regulation of genes
• Most standard image processing algorithms extract the signal
intensities for each spot and from the surrounding background
• The measurement of background intensities can be averaged
over entire arrays or taken from the area adjacent to a spot
• The background intensity derived from the intensity values of
the lowest 2% of cells on the chip, establishes an overall
baseline intensity to be subtracted from all cells before gene
expression levels are calculated

36. • Early days: fixed fold-change cut-off (usually twofold) was
used to define differentially expressed genes
– does not take into account the biological and experimental
variability in the data
– thus many genes with high fold-changes but poor-quality
data were mistakenly identified as being differentially
expressed
– whereas genes with reproducible data but low fold-changes
were missed

37. Expression Ratio
Logarithmic Transformation of Expression Ratio
Log2(expression ratio)
if the expression ratio is 1, then log2 (1) equals 0 represents no change in
expression. If the expression ratio is 4, then log2 (4) equals +2 and for
expression ratio of log2 (1/4) equals -2
For each gene k on the array, where Rk represents the spot intensity metric for the
test sample and Gk represents the spot intensity metric for the reference sample.

38. • sophisticated statistical approaches-involve three steps:
– calculating a test statistic, assigning the significance, and
choosing a cut-off value for the statistical significance
• For a simple two condition experiment
– ordinary t-test, t = M/(s/√n ), where M is the average log-
ratio, s is the standard deviation of the M-values, and n is
the number of replicates
• After a test statistic has been selected, the next step is to
compute the significance (P-value) of the test statistic and to
choose a cut-off value, above which the genes will be
considered as differentially expressed

39. Cluster Analysis
Two Algorithms for Clustering Analysis
• Hierarchical clustering
• K-Means / K-Medians clustering
Expression Data Matrix
• Gene expression data are usually presented in an expression matrix.
Each element is a log ratio. The log ratio is defined as log2 (T/R),
where T is the gene expression level in the testing sample, R is the
gene expression level in the reference sample.
39

40. • Hierarchical Clustering is the most popular method for gene
expression data analysis. In hierarchical clustering, genes with similar
expression patterns are grouped together and are connected by a series
of branches (clustering tree or dendrogram). Experiments with similar
expression profiles can also be grouped together using the same
method.
40

41. • K-Means / K-Medians clustering
41

42. • Microarray has shown great promise
in studying complex diseases such as
cancer. The genome-wide gene
expression profiles of tumor tissues
are considered as the molecular
portraits of various cancers.
• For example, Clustering of breast and
ovarian carcinoma cases is shown in
the figure, 68 breast and 57 ovarian
cases were co-clustered to determine
both similarities and disparities
between the two sample sets.
42

43. Distance
• Applying clustering to expression data is the assumption that genes
with similar expression patterns are likely to be functionally related
• Two most widely used measures are Euclidean distance and
Pearson’s correlation coefficient
• Euclidean distance between two genes G1 and G2 across n samples
• Pearson’s correlation coefficient
• where g1 and g2 are mean expression values for genes G1 and G2
across n sample
Gene A and B are merged first at the level of 1.58. The
position of the splitting point shows the distance between
two genes (or clusters). A low splitting point means short
distance and high similarity.

44. FUNCTIONAL ANALYSIS AND INTERPRETATION
• Generate a list of significant probe sets or probes that are
differentially expressed across experimental conditions
• Add biological knowledge to the selected identifier lists either
from existing literature or from databases such as Entrez Gene,
Unigene, UniProt, Gene Ontology (GO), and Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathways
• Most of these databases classify genes into biological
categories or classes that represent their function
• Estimate the statistical significance of association between the
classes and probes of the obtained list

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The DNA chips are used in many areas as given below:
• Gene expression profiling (Transcriptome profiling)
• Differential expression analysis
• Diagnostics (Detection of SNPs, deletions and duplications) and
genetic engineering
• Analysis of post translational modifications (Alternative splicing
detection)
• Proteomics
• Functional genomics
• DNA sequencing
• Toxicological research (Toxicogenomics)
• Cellular profiling
• Glycome analysis
Applications

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• Provides data for thousands of genes.
• One experiment instead of many.
• Fast and easy to obtain results.
• Different parts of DNA can be used to study gene expression.
ADVANTAGES
• The biggest disadvantage of DNA chips is that they are expensive
to create.
• The production of too many results at a time requires long time for
analysis, which is quite complex in nature.
• The DNA chips do not have very long shelf life, which proves to
be another major disadvantage of the technology.
•Identify gene expression of only those who already reported.
Disadvantages:

47. 47

48. 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
examined tissue.
Use housekeeping genes and positive controls to
ascertain proper functioning of the system.
High background, weak specific signals.
Poor labelling reaction. Check success of labelling reaction specific signals.
before hybridization. 48
TROUBLESHOOTING MICROARRAY EXPERIMENTS

49. 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
labelling reaction.
Use less CyDye nucleotide.
Bubble effect on slide
Air has been trapped under coverslip Remove air bubbles from hybridization
49
TROUBLESHOOTING MICROARRAY EXPERIMENTS

50. Background is higher on one side of slide
50
Air trapped under coverslip
Speckled background on slide b/c CyDye
nucleotides remain in probe
Uneven fluoroscent background on slide

51. 51
Poor slide quality
Low or undetectable Cy3 and/or Cy5 signal.

52. • Infection of chickens with highly pathogenic avian influenza (HPAI)
H5N1 virus leads to 100% mortality within 1 to 2 days but in ducks
causes mild or no clinical signs.
• The rapid onset of fatal disease in chickens, but not in ducks, suggests
underlying differences in their innate immune mechanisms.
• Chicken Genechip microarrays (Affymetrix) use to analyse the gene
expression profiles of primary chicken and duck lung cells infected
• with a low pathogenic avian influenza (LPAI) H2N3 virus and two HPAI
H5N1 virus subtypes.
• Pro-inflammatory cytokine genes, interleukin (IL)- 6, IL-8 and IL-10
were highly up-regulated in both H5N1 virus infected chicken cells; in
contrast, IL-8 expression was unchanged, and IL-6 and IL-10 were down
regulated in infected duck cells with the same viruses.
• Expression of IL-18 was up-regulated in duck cells but was down-
regulated in chicken cells
52

53. • Chicken Genome Arrays were used to construct an adipose tissue gene
expression profile of 7-week-old broilers, and to screen adipose tissue
genes that are differentially expressed in lean and fat lines divergently
selected over eight generations for high and low abdominal fat weight.
• 230 genes that were differentially expressed between the two lines were
screened out; these were mainly involved in lipid metabolism, signal
transduction, energy metabolism, tumorigenesis and immunity.
• Subsequently, real-time RT-PCR was performed to validate fifteen
differentially expressed genes screened out by the microarray approach
and high consistency was observed between the two methods.
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