Transcript Bild 1 - Sina Med Nano

Microarray
DNA
Why DNA?
• 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
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.
Microarray
• 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/]
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 ,...
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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
Slide
subarray
spot
Gene/Protein
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 surfaces
-P -P -P
1.Poly-L-lysine
+
+
NH3 NH3
-P + -P -P+
NH3
NH3
2.Amin
NH2
CH
3.Aldehyde
4.Epoxy
NH2
O
CH2
CH
N
=
=
O
CH
NH
CH2
HO CH
+ H2O
Robot Spotting
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Spotting of DNA library on slides
Microtiter plates
PCR products
from >9000 genes
Glass slides
3000 spots per slide
InkJet (HP/Canon) technology
Cartoon of Printing Process
(side view from the table top)
Plate with wells
containing probes
microarray slides
vacuum
wash
station
Dip pen Nanolithograph
Future
Today
3 μm
Tomorrow
3 μm
Micro scale array
Nano scale array
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(cont)
•Electrochemical Sensing
•Principle
•Oxidation/Reduction
eee-
“Electrons flow from the Au
Electrode to intercalated MB+ and
Then are accepted by the Fe(CN)64-”
E.M. Barton, J.K., N.M. Hill, M.G (1999) Nucleic
Acid Research 27, 4830.
e-
Proposed Chip Concept
•Combination of Biological and Electrical chips
eee-
Circuitry
Nano DNA Array
A
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 a
List 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
Induced
Expressed in
both
conditions
Repressed
Merged
images
R
G
Expression ratio - Normalization
∆ Gene expression
R
G
600
1200
0.07
Test/ Experimental
G
Reference/ Control
T=
5600
17500 16500 13500
0.03
R
0.4
11600 13000 15500 18000
10900 6500
1.0
2.0
2500
800
6.2
22.5
0/0
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
Using cDNA Microarrays to Measure
mRNA Levels
Microarray Slide
ACCTG...G
ACCTG...G
ACCTG...G
TTCTG...A
TTCTG...A
TTCTG...A
GGCTT...C
GGCTT...C
GGCTT...C
ATCTA...A
ATCTA...A
ATCTA...A
ACGGG...T
ACGGG...T
ACGGG...T
CGATA...G
CGATA...G
CGATA...G
Sample 1
Spots
(Probes)
Unknown
mRNA
Sequences
(Target)
Sample 2
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Extract mRNA
Sample 1
ACCTG...G
ACCTG...G
ACCTG...G
TTCTG...A
TTCTG...A
TTCTG...A
GGCTT...C
GGCTT...C
GGCTT...C
ATCTA...A
ATCTA...A
ATCTA...A
Sample 2
ACGGG...T
ACGGG...T
ACGGG...T
CGATA...G
CGATA...G
CGATA...G
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Convert to cDNA and Label with
Fluorescent Dyes
Sample 1
??????????
??????????
??????????
??????????
TTCTG...A
TTCTG...A
TTCTG...A
??????????
ACCTG...G
ACCTG...G
ACCTG...G
??????????
??????????
??????????
??????????
??????????
GGCTT...C
GGCTT...C
GGCTT...C
ATCTA...A
ATCTA...A
ATCTA...A
Sample 2
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
CGATA...G
CGATA...G
CGATA...G
??????????
ACGGG...T
ACGGG...T
ACGGG...T
??????????
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Mix Labeled cDNA
Sample 1
??????????
GGCTT...C
GGCTT...C
GGCTT...C
ATCTA...A
ATCTA...A
ATCTA...A
??????????
TTCTG...A
TTCTG...A
TTCTG...A
??????????
ACCTG...G
ACCTG...G
ACCTG...G
??????????
??????????
Sample 2
??????????
??????????
??????????
??????????
CGATA...G
CGATA...G
CGATA...G
??????????
ACGGG...T
ACGGG...T
ACGGG...T
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Hybridize cDNA to the Slide
Sample 1
TTCTG...A
TTCTG...A
TTCTG...A
GGCTT...C
GGCTT...C
GGCTT...C
ATCTA...A
ATCTA...A
ATCTA...A
ACGGG...T
ACGGG...T
ACGGG...T
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
ACCTG...G
ACCTG...G
ACCTG...G
Sample 2
CGATA...G
CGATA...G
CGATA...G
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Excite Dyes with Laser
Sample 1
ACCTG...G
ACCTG...G
ACCTG...G
??????????
??????????
??????????
??????????
GGCTT...C
GGCTT...C
GGCTT...C
??????????
??????????
??????????
??????????
ACGGG...T
ACGGG...T
ACGGG...T
TTCTG...A
TTCTG...A
TTCTG...A
ATCTA...A
ATCTA...A
ATCTA...A
Sample 2
CGATA...G
CGATA...G
CGATA...G
48
??????????
??????????
Scan
Sample 1
ACCTG...G
ACCTG...G
ACCTG...G
??????????
??????????
??????????
??????????
GGCTT...C
GGCTT...C
GGCTT...C
??????????
??????????
??????????
??????????
ACGGG...T
ACGGG...T
ACGGG...T
TTCTG...A
TTCTG...A
TTCTG...A
ATCTA...A
ATCTA...A
ATCTA...A
Sample 2
CGATA...G
CGATA...G
CGATA...G
49
??????????
??????????
Quantify Signals
Sample 1
ACCTG...G
TTCTG...A
7652
138
5708
4388
GGCTT...C
ATCTA...A
8566
765
1208
13442
ACGGG...T
CGATA...G
6784
9762
67
239
Sample 2
50
Comparative genomic hybridization
CGH array
Chip-on-chip
Tiling array
Partially overlapping probes
Non overlapping probes
Genome Tiling Arrays
800 bp
25-36mer
earray
Design a custom microarray
http://earray.chem.agilent.com/
MIAMI
Minimum Information About a Microarray Experiment
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
Illumina BeadArray
Miller M B , Tang Y Clin. Microbiol. Rev. 2009;22:611-633
Illumina Bead Arrays
• Oligonucleotides (50mers) immobilized on
glass beads
• Identifier tag on each
oligo
• Usually ~ 30 beads per
probe
Protein arrays
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
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.
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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.
Different type arrays
Protein-protein array
Small molecule array
Protein-activity array
Methods of synthesis
• Printing
• 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)
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)
Integrated Microarray-Microfluidics
-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
SNP-array and gene expression DNA microarray data
mapped to chromosomal positions.
Use of SNPscanPlot to visualize chromosomal
anomalies in apparently normal individuals.