Professional Documents
Culture Documents
and Applications
Purnima Kartha. N
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INTRODUCTION
• A microarray consists of a solid surface to which biological molecules are
arranged in a regular pattern.
• Applicable in the fields of DNA, proteins, peptides and small molecules like
metabolites and drugs.
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DNA MICROARRAY
• Orderly arrangement of thousands of
identified sequenced genes printed on an
impermeable solid support, usually glass,
silicon chips or nylon membrane.
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• Measurement of Gene Expression.
PRINCIPLE
Hybridization between two DNA strands
Microarrays use relative quantitation : Intensity of a feature is compared to
the intensity of the same feature under a different condition, and the identity
of the feature is known by its position.
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TYPES OF MICROARRAYS
1. Glass DNA microarrays which involves the
micro spotting of pre-fabricated cDNA fragments
on a glass slide.
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1. ARRAY PRINTING
• The microarray slide is uniformly coated with a chemical compound that
will interact with and immobilize nucleic acids, irreversibly binding them
to the surface.
• Nucleic acids are deposited on the slide by contact printing, and treated
with UV light or baking at 80°C to crosslink them to the slide surface.
• The printed slides are stored desiccated at room temperature, in the dark,
until required for experimentation.
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The selection of probe sequences for a
PROBES microarray design depends upon the use
envisaged for the array.
Specific probes can be designed against genes,
transcripts or portions of transcripts.
1. Clone sets
2. cDNA library preparation
3. Specific DNAs amplified.
All clones purified by gel filtration/
precipitation to reduce unwanted salts.
Selection of probes requires balancing of 4
criteria during their production: Sensitivity,
Specificity, Noise, and Bias. 8
Printing PCR products onto glass slides:
• Printing involves the sequential transfer of individual PCR products from
the plates to defined areas of glass slides.
• Glass slides pre-coated with poly lysine, amino silanes or amino-reactive
silanes - to increase the hydrophobicity of the slide, improving the
adherence of the deposited DNA and minimising spreading.
• Few nLs of each DNA is deposited onto each slide, resulting in formation
of spots of 50-150µM diameter.
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Printing of cDNA microarrays
Print-tips collect
cDNA from wells
96-well plate
Contains cDNA
probes
Print-tip
cDNA clones
Glass slide
Array of bound cDNA probes 10
Printing of High-density oligonucleotide microarrays
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Post Processing of slides:
• DNA is usually cross-linked to the glass slides by treating with UV light
or baking at 80°C, and residual amines are blocked be reaction with
succinic anhydride.
• The printed slides are stored desiccated at room temperature, in the dark,
until required for experimentation.
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2. SAMPLE PREPARATION
In sample preparation, RNA from the host organism is isolated,
converted to cDNA and labelled with dyes before hybridization to the
array.
1. RNA extraction from the tissue of interest.
Quantity, and integrity of the total or mRNA used is important
By using dNTPs that have dye molecule directly coupled to the base, with
cyanine 3-dCTP (Cy3-dCTP) and cyanine 5-dCTP (Cy5-dCTP).
Conjugates of the Alexa dyes, Alexa555 and Alexa647, the spectral
analogues, fluorescent and photo-stable and are therefore the most
commonly used alternatives.
.
Ad: Quick and simple, requiring relatively few steps, and therefore are easy to
scale up for high throughput.
Disad: Require high amount of RNA (approx 25–100 mg total RNA) for
labelling reaction. The bulky dye-coupled nucleotides reduce the efficiency of
the reverse transcriptase and lead to dye bias. 15
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Indirect labelling:
The RT enzyme incorporates an amino-allyl dNTP into the cDNA instead of
Cy-dCTP.
1. Aminoallyl-dNTP is added to ntd mix in the RT reaction to produce first strand
cDNA
2. After first strand synthesis, an amine-reactive Cy dye is chemically coupled to
the aminoallyl groups, thus labelling the cDNA.
The CyDyes have NHS (N-Hydroxysuccinimide) esters that react with the
aminoallyl groups of the cDNA.
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3. MICROARRAY HYBRIDISATION
During the hybridization reaction, labelled targets interact with the tethered probes
due to sequence complementarity.
After-Hybridisation Washing:
To remove unbound target and any target loosely bound to imperfectly
matched sequences.
For good quality arrays, it is essential that both hybridization and washing is
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uniform across the array and that the surface is evenly dried before scanning.
4. DATA ACQUISITION &ANALYSIS
• Gene expression levels are evaluated by measuring the amount of reference
and test probe that binds to each arrayed cDNA.
• Fluorescence is detected on arrays by means of a scanner or reader and
saved as a digital image.
• These data are then imported into software that converts the fluorescence
into pixels that can be counted.
• Following background subtraction and normalisation, expression ratios can
be calculated.
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1. Scanning
o A microarray scanner uses a light source to excite the fluorophores present on
the sample molecules and then detects the emitted light, which is most
typically stored as a 16-bit tiff (tagged image format) files for each
wavelength scanned.
o Each image is composed of a matrix of pixels where each pixel represents the
fluorescence intensity of a small area of the array.
o This digital image represents the ‘raw data’ from which the fluorescent signal
of each array element will subsequently be quantified and the expression level
of each target inferred.
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Detector
PMT
Image
Cy5: 635nm
Cy3: 532nm
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Scanners, according to the type of excitation and detection technology they
utilize. Scanner
s
Laser CCD based
Scanners Scanners
Laser Scanners:
• Use narrowband laser illumination to excite
fluorophores and capture the resulting
fluorescence with a PMT detector.
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APPLICATIONS OF DNA
MICROARRAY TECHNOLOGY
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GENE EXPRESSION MONITORING
Measurement of absolute levels of expression for each represented gene by
quantifying the amount of targets that hybridise with the arrayed probes.
Microarray targeting the 16S rRNA gene developed for the detection of a
panel of 40 predominant human intestinal bacterial pathogens in human fecal
samples.
Rapid diagnosis of bloodstream infections caused by common bacterial
pathogens in the paediatric and general populations.
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SNP arrays have been used is the mapping of human disease susceptibility
loci as published in GWAS. In order to facilitate the GWAS, a detailed
human haplotype map has been created using over a million SNP (The
International HapMap Consortium 2005).
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PROTEIN MICROARRAYS
Protein microarray is an emerging technology that provides a versatile
platform for characterization of hundreds of thousands of proteins in a
highly parallel and high-throughput way.
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PROTEIN FUNCTION ARRAYS
Developed for the study and elucidation of the function and interaction of
various biological molecules.
Constructed using individually purified proteins.
They enable the study of various biochemical properties of proteins such
as:
Binding activities and structure elucidation of proteins
Interactions of proteins with proteins, DNA, lipids, drugs, peptides etc.
Post translational modifications.
enzyme-substrate relationships
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ARRAY FABRICATION
Protein arrays have been made of a miniaturised solid support spotted with
a protein, peptide or antibody.
Immobilization of the proteins to the array surface via chemical (glass
arrays), hydrophobic (hydrogels) or electrostatic (nitrocellulose)
interactions.
Glass arrays are mostly coated with aldehyde or BSA-NHS, which react
eith the primary amines on the N-termini or side chains of proteins.
Protein and peptide arrays can be spotted/inkjet printed, or peptide arrays
can be synthesized in situ on a solid substrate.
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SAMPLE LABELLING, HYBRIDISATION,
AND DETECTION
Fluorescence labelling of the proteins or antibodies with Cy3 and Cy5,
FITC(green), Rhodamine(Red) and Texas Red.
Mono-functional NHS esters of CyDyes can be used to label amine
residues in antibodies and other proteins, or maleimide esters of CyDyes
can be used to label free thiol groups.
Hybridization conditions for protein arrays are similar to those used in
ELISA assays.
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SAMPLE LABELLING, HYBRIDISATION,
AND DETECTION
The array incubated with the solution containing the protein(s) under
conditions that promote binding between the immobilized probe and the
target molecules.
Washed under conditions that remove weakly binding target molecules.
Binding/interactions are then detected and quantified using appropriate
scanners.
Detection of binding to the array can then be quantified using a standard
DNA microarray scanner.
Another method of detection is by the use of a secondary labelled
antibody directed to the target protein. 53
APPLICATIONS
Major areas where protein arrays can potentially be used:
Proteomics
Functional analysis
Diagnostics
3. Drug Targets
Entire proteome printed on a chip used to study drug interactions.
4. Autoimmune Diseases
Charpin et al. used ProtoArray (Invitrogen), a protein Microarray with a
nitrocellulose layer where 8268 human proteins with a GST-Tag are spotted, to
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detect specific marker for early stages of RA (WIBG, GABARALP2 und ZNF706).
5. Protein–small molecule interaction
Detection of small molecules in case of drug abuse or doping in sports.
Du et al. developed a Protein Microarray for the detection of different prohibited
drugs in various biological fluids (e.g., whole blood, serum, urine and oral fluid).
These arrays have been used for the detection of different drugs of interest including
morphine and meprobamate in blood.
6. Allergen arrays
Many allergens have the same protein epitopes, which means that the same
sIgE antibody can bind to different allergens from different sources.
Protein Microarrays or Bead based Systems, can check for cross-reactivity of
allergens in one assay.
Lower costs, lower sample volume and the possibility of measuring a lot more 56
components in one experiment.
TECHNICAL CHALLENGES TO PROTEIN
MICROARRAYS
Retaining the secondary and tertiary structure, and therefore activity on
the microarray;
Identifying and isolating an agent with which to capture the proteins of
interest.
Accurately measuring the degree of protein binding with a system that is
both sensitive and with a wide dynamic range.
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CARBOHYDRATE/GLYCAN ARRAYS
Not as widely used as DNA or protein arrays.
Isolation of carbohydrates from natural sources requires multiple and
varied purification steps.
Chemical synthesis of carbohydrates/glycans requires multiple selective
protection and deprotection steps during synthesis of oligosaccharides,
because of the often three-dimensional nature of the branched linkages in
oligosaccharides.
Carbohydrates have a low mass and are mainly hydrophilic, it is difficult to
directly immobilize them onto solid matrices by noncovalent bonding.
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CARBOHYDRATE/GLYCAN ARRAYS
Immobilization of carbohydrate probes by spotting and immobilization
onto nitrocellulose membranes, by linkage of oligosaccharides to lipids,
which were immobilized to nitrocellulose, or by immobilization to
hydrophobic polystyrene slides.
Immunostaining of the carbohydrate arrays or detection of binding using
labelled secondary antibodies have been used to detect binding to
carbohydrate arrays.
Labeling of free carbonyl groups on glycoproteins and carbohydrates can
also be achieved, using CyDye™ hydrazides,
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APPLICATIONS
Used both as ‘capture’ arrays to detect the expression of a particular
biological molecule such as an antibody, and as assays for functional
interaction studies, such as for studying the specificity of antibodies.
Thirumalapura and co-workers have constructed lipopolysaccharide (LPS)
microarrays, (LPS from E. coli O111, E. coli O157, and S. typhimurium).
They have used these arrays both to monitor the specificity of MAbs.
Detection of bacterial toxins using immobilized N-acetyl galactosamine
(GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives.
Use of lectin arrays to profile cell surface carbohydrate expression
(mannose, galactose, and GlcNAc expression) in mammalian cells (Zheng60
et al., 2005).
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REFERENCE
Microarray and its applications: Rajeshwar Govindarajan, Jeyapradha Duraiyan, Karunakaran Kaliyappan,
Murugesan Palanisamy
Protein microarray technology: Markus F. Templin, Dieter Stoll, Monika Schrenk
Protein microarray technology: David A Hall, Jason Ptacek.
DNA microarrays: Types, Applications and their future: Roger Bumgarner
Applications of DNA Microarray in Disease Diagnostics: Yoo, Seung Min, Jong Hyun Choi, Sang Yup Lee, and Nae
Choon Yoo
Protein Microarrays: Chien-Sheng Chen Heng Zhu
Microarray Technology in Practice : Steven Russell, Lisa A. Meadows and Roslin R. Russell
Microarray Technology Through Applications: Francesco Falciani
Bioarrays- From Basics to Diagnostics: Krishnarao Appasani
Microarray Bioinformatics: Dov Stekel
An Introduction to Microarray Data Analysis: M. Madan Babu
Microarray technology Ágnes Zvara, Klára Kitajka, Nóra Faragó, László G. Puskás 62
DNA Microarray Technology: Devices, Systems, and Applications: Michael J. Heller
THANK YOU
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