microarray. dna dna can be manipulated to create exact copies that are extremely accurate. dna is...

Microarray

• DNA can be manipulated to create exact copies that are extremely accurate.

• DNA is predictable and programmable.

• DNA also has the ability to store enormous amounts of information

Why DNA?

DNA chips

• Based on genetic information • Based on DNA structure

What is Microarray ?

• Microarrays are fabricated by high-speed robotics, generally on glass but

sometimes on nylon substrates, for which probes with known identity are

used to determine complementary binding, thus allowing massively

parallel screening studies.

• An experiment with a single DNA chip can provide researchers

information on thousands of genes simultaneously - a dramatic increase in

throughput.

• In 1975, Ed Southern proved the concept that DNA fixed to a solid support could be used to attract complementary DNA strands in a process now known as Southern blotting.

• In 1991, Stephen Fodor at Affymax reported the fabrication of DNA microarrays on the surface of glass chips by combining the photolithographic method

• In 1993, he co-founded Affymetrix, in order to develop microarrays with hundreds of thousands of different oligonucleotides.

• Next year Affymetrix commenced manufacturingand selling its first DNA microarray, GeneChip, and the DNA microarray market was born.

• Pat Brown at Stanford University developed a method for mechanically arraying and immobilizing numerous PCR-derived cDNAs using a robot to print onto simple glass slides. [http://brownlab.stanford.edu/]

Microarray

Types of Arrays – Applications:

• DNA Microarrays:

Expression profiles, disease research (cancer), DNA sequencing, mutation analysis, gene discovery, diagnosis, drug discovery,…

• RNA Microarrays:

RNA-protein interactions, biological function of proteins, drug discovery,…

• Protein Microarray (chips):

Enzyme profiling, Protein-protein Interaction, Protein-ligand interaction ,...

8

Arrays

• Complementary DNAs (cDNA)• Amplicons• Oligonucleotides• Proteins

Arrangged on

• Nylon / Nitrocellulose• Glass • Silicon

Probe production

Oligos (up to 60-80 nt)-synthesized on silica (Affymetrix)-synthesized and spotted, or sprayed (Agilent)

PCR products (100-500 nt)-produced by PCR from plasmid templates-purified and spotted

cDNA (1-5 kB)-clones expanded by bacterial cultures (1000s)-plasmid purified-spotted to substrate (nitro, plastic, glass)

DNA chip classification

• Low density 10-1000 probes for diagnosis• Medium density 1000-10000 probe for mutation analysis• High density 10000-? probes for gene sequencing

Microarray parameters

subarray

Slide

Gene/Protein

spot

Selected parameters of DNA chips

Array production

• In silica synthesis- Affymetrix (photomasking)- Nimblegen- density unlimited (>500K features/cm2)

• Spotting- robotic ‘quills’ or ‘loop and pin’- density limited by diffusion properties of

solution and substrate (16-32K/slide)• Spraying

- nano/picoliter sprays (inkjet, Agilent-H/P)

Affymetrix Photolithography

Febit/NimbleGen photolitography

Slide surfacesP P P- - -

NH3 NH3

+ +NH3 NH3

+ +P P P- - -

1.Poly-L-lysine

2.Amin

3.Aldehyde

4.Epoxy

CH

=

ONH2 CH

=

N +H2O

CH

CH2 O

NH2

NH

CH

CH2

HO

Robot Spotting

Spotting of DNA library on slides

Microtiter plates Glass slides

3000 spots per slidePCR products

from >9000 genes

InkJet (HP/Canon) technology

Plate with wellscontaining probes

microarray slides

vacuumwashstation

Cartoon of Printing Process)side view from the table top(

Dip pen Nanolithograph

Micro scale array Nano scale array

Today Tomorrow

3 μm 3 μm

Future

Process of the Microarray experiment

Detection methods

•Radioactive- incorporate 32P-CTP into RT step- sensitive, but nearly obsolete

•Fluorescent- single color, or 2 color (Cy3, Cy5)- 2 color allows 2 samples on 1 chip

•Antigenic- hybrid capture uses antibody to Double strand

•Electrochemical- circuit sensing

•Electrochemical Sensing•Principle•Oxidation/Reduction

e-

“Electrons flow from the AuElectrode to intercalated MB+ andThen are accepted by the Fe(CN)6

4-”E.M. Barton, J.K., N.M. Hill, M.G (1999) NucleicAcid Research 27, 4830.

e-

e-

e-

Electrochemical Sensing(cont)

•Combination of Biological and Electrical chips

Proposed Chip Concept

Circuitry

A

e-

e-

e-

Nano DNA Array

http://micro.magnet.fsu.edu/chipshots/cyrix/486dx2diemedium.html

Hybridization

GeneTAC Hyb station

Image processing by Laser scanning

The microscope slide containing the microarray is placed inside a microarray scanner, where the slide is scanned with two lasers to detect the bound green and red cDNAs.

Microarray Data Analysis

How to Handle Microarray Data?

• Signal Generation from Image

• Normalization

• Filtering

• Statistical Tests

• Clustering

The Main Goal of Microarray Data Analysis is to Generate aList Of ‘Interesting’ Genes

Image Analysis

1. Gridding: identify spots (automatic, semiautomatic, manual)

2. Segmentation: separate spots from background

3. Intensity extraction: mean or median of pixels in spot

4. Background correction: local or global

Image Analysis

• Spot Finding

• Background subtraction

• Intensity Calculation

Background subtraction

● Global background

● Local background

● Negative control

● Morphological opening

Data analysis

What accounts for the varying colors?

These actually correspond to the amount of cDNA that binds to the complementary strands on the spot.

Data analysis

Merged images

Induced RepressedExpressed in

both conditions

R G

http://cropwiki.irri.org/gcp/index.php/Image:Induction-repression.GIF

Expression ratio - Normalization

R

GT=

Reference/ Control

Test/ Experimental

1800011600 13000 1550056001200600

8002500650010900135001650017500

22.56.22.01.00.40.070.03

R

G

0/0

∆Gene expression

Reporting your results

Microarray

Gene

The expression ratios for every gene can be organized into a table where each column is a microarray and each row is a gene. This representation however is overwhelming in experiments involving thousands of genes and data.

Gene expression microarray

43

Using cDNA Microarrays to Measure mRNA Levels

ACCTG...GACCTG...GACCTG...G

TTCTG...ATTCTG...ATTCTG...A

GGCTT...CGGCTT...CGGCTT...C

ATCTA...AATCTA...AATCTA...A

ACGGG...TACGGG...TACGGG...T

CGATA...GCGATA...GCGATA...G

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Sample 1

Sample 2

Microarray Slide

Spots(Probes)

UnknownmRNA

Sequences(Target)

44

Extract mRNA







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Sample 1

Sample 2

45

Convert to cDNA and Label with Fluorescent Dyes







Sample 1

Sample 2

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Sample 1

Sample 2

46

Mix Labeled cDNA







Sample 1

Sample 2??????????

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47







Sample 1

Sample 2

Hybridize cDNA to the Slide

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48







Sample 1

Sample 2

Excite Dyes with Laser

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49







Sample 1

Sample 2

Scan

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50

Quantify Signals

ACCTG...G

76527652138138

TTCTG...A

5708570843884388

GGCTT...C

85668566765765

ATCTA...A

120812081344213442

ACGGG...T

6784678497629762

CGATA...G

6767239239

Sample 1

Sample 2

Comparative genomic hybridization CGH array

Chip-on-chip

Tiling array

Partially overlapping probes

Non overlapping probes

Genome Tiling Arrays

800 bp

25-36mer

earrayDesign a custom microarray

http://earray.chem.agilent.com/

http://earray.chem.agilent.com/

http://www.mged.org/Workgroups/MIAME/miame.html

•Array Design•Experimental Design

•Samples used, extract preparation and labelling •Hybridization procedures and parameters

•Measurement data and specifications of data processing

MIAMIMinimum Information About a Microarray Experiment

http://www.mged.org/Workgroups/MIAME/miame.html

Illumina BeadArray

Miller M B , Tang Y Clin. Microbiol. Rev. 2009;22:611-633

Illumina Bead Arrays• Oligonucleotides (50-

mers) immobilized on glass beads

• Identifier tag on each oligo

• Usually ~ 30 beads per probe

The promise of the protein microarray is the ability to interrogate a large number of proteins simultaneously in a high-density format for disease diagnosis, prognosis or efficacy of therapeutic regime as well as for biochemical analysis

Protein arrays

A new tool on behalf of traditional macroscopic technology such as 2 dimensional electrophoresis, mass spectroscopy (MS), capillary electrophoresis (CE), and enzymelinked immunosorbent assay (ELISA)

The protein chip, although analogous to DNA chip, faces much great challenges in terms of commercial product. This systematic analytical device for proteome study requires biological surface fabrication to retain the activity of immobilized protein, miniaturization of protein array, and detection technology with high sensitivity.

Why study proteins?

• They are the machines that make cells function.

• RNA levels do not always accurately predict protein levels.– Often processes are regulated at the transcriptional level.– Some processes are controlled post-transcriptionally.

• Proteins are the targets of drugs.

Protein-protein array

Different type arrays

Protein-activity array

Small molecule array

Methods of synthesis

• In-situ synthesis

• Nucleic acid programmable protein array (NAPPA) • Protein in situ array (PISA)

• In situ puromycin-capture

• Nano-well array format

• DNA array to protein array (DAPA)

• Printing

Nucleic acid programmable protein array (NAPPA)

Protein in situ array (PISA)

In situ puromycin-capture

Nano-well array format

DNA array to protein array (DAPA)

-Perform synthesis without pre-assembly amplification

-Enables increased utilization of high-density DNA microarrays by:

-reducing pool complexity-limiting undesired oligo interactions

-maintaining reagent concentrations at desired levels

Integrated Microarray-Microfluidics

SNP-array and gene expression DNA microarray data mapped to chromosomal positions.

Use of SNPscanPlot to visualize chromosomal anomalies in apparently normal individuals.

microarray. dna dna can be manipulated to create exact copies that are extremely accurate. dna is...

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