DNA Microarrays
What is a microarray
A surface on which sequences from
thousands of different genes are
covalently attached to fixed locations
(probes).
Glass slides
Silicon chips
Utilize the selective nature
of DNA-DNA or DNA-RNA
hybridization
Types of microarrays
Probe Manufacture
Detection
Spotted Arrays
DNA probes are synthesized Two-color detection
and then “spotted” onto the Spotted Array: Compare two
microarray samples by labeling with two
Customized for each different flurophores and
experiment “in-lab” analyzing on the same array
Low cost Cy5 (red) and Cy3 (green)
Oligonucleotide
microarrays One-color detection
DNA oligonucleotides are Determine gene expression
synthezised directly on the
level
microarray
Commercial arrays One array per sample
(Affymetrix, Nimblegen and
Combimatrix)
expensive
Phosphoramidite chemistry
Stable = can actually be shipped
High coupling yield
Synthesis on a solid support
Monomer addition
Still a manual method, not consistent
Faster cycle time of 20 minutes
Using some nasty reagents (e.g. DMAP, DMF,
Thiophenol)
Very cyclical and repetitive = not fun
Coupling efficiency
100 %
Phosphoramidite
chemistry at 98%
50 % coupling efficiency
0%
10 bases 20 bases
Solid Phase Synthesis
Controlled Pore Glass (CPG) support covalently
linked with one of the four nucleosides
Reactive groups of nucleosides are blocked or
protected to prevent unwanted side reactions
DNA synthesis occurs by connecting nucleoside
monomers one at a time to the 5' end of growing
chain (3' to 5' end)
DNA chain is cleaved from the support by ammonia
treatment
Solid Phase Synthesis
CPG support covalently linked with one of the four
nucleosides
DMT - protected nucleoside
CH 3
O
BASE
C O
O O
C CH CH2 C NH CH CH CH S O
2 2 2 2
O i
O O O
CH3
Solid Phase Synthesis
CPG support covalently linked with one of the four
nucleosides
Reactive groups of nucleosides are blocked or
protected to prevent unwanted side reactions
Protection of Base Amino Groups
O
O NH N
NH C N
HN N
N N
C O
N N CH
CH3 CH3
O
O
NH C
HN CH 3
N
O N O N
Solid Phase Synthesis
CPG support covalently linked with one of the four
nucleosides
Reactive groups of nucleosides are blocked or
protected to prevent unwanted side reactions
DNA synthesis occurs by connecting nucleoside
monomers one at a time to the 5' end of growing
chain (3' to 5' end)
Direction of synthesis
Oligonucleotide
5’ 3’ CPG
Natural DNA
5’ 3’
Solid Phase Synthesis
CPG support covalently linked with one of the four
nucleosides
Reactive groups of nucleosides are blocked or
protected to prevent unwanted side reactions
DNA synthesis occurs by connecting nucleoside
monomers one at a time to the 5' end of growing
chain (3' to 5' end)
DNA chain is cleaved from the support by ammonia
treatment
Typical quantities of oligonucleotide
obtained from different scales
Synthesis scale Crude Yield (O.D.) (20 mers)
Primer (40 nmole) 5 - 10
0.2 umole 20 - 25
1 umole 100 - 120
10 umole 800 - 1000
Analysis & Purification
PAGE
HPLC
CE (capillary electrophoresis)
OPC (oligonucleotide Purification Cartridge)
Quantitation & Storage
UV spectroscopy: Measuring OD at 260 nm
1 OD 260 for ssDNA = 33 ug/ml
Storage: stable with little or no degradation for
long periods of time (over a year)
Alternative chemistries
Antisense (phosphorothioate)
RNA oligoribonucleotides
(using 2' silyl 5' DMT-CE phosphoramidite RNA monomers)
5' modified oligonucleotides
5' modified oligonucleotides
Aminolink
Enzyme - labeled
Biotin - labeled
Fluorescein-lebeled
Microarray Platforms
Spotted
DNA fragments (usually created by PCR)or oligos are stuck to glass slides
The size of the fragment can be any length (usually 500 bp-1 kb)
The size of the oligos range from 20-100 nts
These arrays can be created in individual labs using “affordable” equipment
Affymetrix
Affymetrix arrays are typically limited to oligos of 20-25 nts
The probes on these arrays are synthesized using a light mask technology
Photo-sensitive reactions are used to remove a blocking group and then extend
It is very costly to fabricate masks for a new array design
Not commonly used for custom arrays
Types of Microarray Platforms
NimbleGen
Maskless Array Synthesizer technology uses PowerPoint projector parts; 786,000 tiny
aluminum mirrors are used to shine light in specific patterns,
Photo deposition chemistry allows single nt extensions.
380,000 or 2.1 million oligos/array (50mers).
Agilent
Uses ink-jet printer technology.
Grows another base at the end of a molecule by deprotecting the end and
attaching a new base with a protected end to avoid duplication.
244,000 oligos/array (60mers).
Combimatrix
Semiconductor technology directs the assembly of a specific sequence of DNA bases in
response to a digital command. Each feature on the array (a microelectrode) is digitally
addressed to controls the addition of a new base. 12,000 oligos (50mers)/array; 40,000
feature arrays in development.
Making a Spotted Arrays
Probes
cDNA microarrays (up to 3000 bp)
Long-oligonucleotide spotted arrays: uniform
length (20 -100 bp)
Customized spotted array
Spotting pins draw fluid (containing DNA
probes) by capillary action and form spots on
the side through surface tension interaction
between the surface and spotting buffer.
“spotted” onto glass slide using robotic arms
Commercial spotted arrays (Agilent)
SurePrint Technology
Iike an inkjet printer, prints sets of cDNA
clones or oligonucleotides one-by-one
Spotted Arrays: Sample Preparation and Two-color Detection
Allows a direct
comparison
between two
different samples
DNA microarrays can be manufactured
by:
• Photolitography (Affymetrix, Nimblegen)
• Inkjet (Agilent, Canon)
• Robot spotting (many providers)
Affymetrix photolitography
• Each probe 25 bp long
• 22-40 probes per gene
• Perfect Match (PM) as
well as MisMatch (MM)
probes
Masked Array Synthesis (Affymetrix)
Maskless Arrays (Nimblegen)
NimbleGen photolitography
Robot Spotting
InkJet (HP/Canon) technology
Making a Oligonucleotide array
Probes
20-100 bp oligonucleotide probes
Electrochemistry - CombiMatrix Array
Each microelectrode selectively generates chemical
reagents by electrochemical reaction.
Controls the building of DNA on a semiconductor chip by the
software-controled turning on and off of electrodes.
Photolithography - Affymetrix Array
manipulates light to direct the chemical synthesis of the probes
Mask directs the flow of U.V. light
Uses light sensitive protecting
groups.
Applications
Expression profiling
Comparative genomic hybridization
Single nucleotide polymorphism
Sequencing
Summary
References
GeneChip Expression Analysis Technical Manual. http://www.affymetrix.com/
support/downloads/manuals/expression_analysis_technical_manual.pdf.
McInnerney Kate. One-cycle eukaryotic target prep protocol.
http://www.homepage.montana.edu/~kmcinner/Affyinfo.html
Agilent SurePrint Technology (2003) Retrieved March 23, 2008 from
http://www.chem.agilent.com/temp/radD1958/00000176.PDF
Spotted Array Printing Techniques
http://www.flychip.org.uk/printingintroduction.php
NCBI A Science Primer: Microarrays: Chipping away at the myteries of science
and medicine. www.ncbi.nlm.nih.gov/About/primer/microarrays.html
Affymetrix GeneChip Workflow tools
http://www.invitrogen.com/content.cfm?pageid=11455
Affymetrix Educator Resources
http://affymetrix.com/corporate/outreach/educator.affx
Image Analysis
1. Gridding: identify spots (automatic,
semiautomatic, manual)
2. Segmentation: separate spots
from background. Fixed circle (B),
Adaptive circle C, Adaptive shape
(D), Histogram
3. Intensity extraction: mean or
median of pixels in spot
4. Background correction: local or
global
Distribution of mRNA levels in different cells
Photolithography
Removal of protecting groups and caps
DNA Synthesis Chemistry Cycle
Detritylation
Coupling
Capping
Oxidation
Oligonucleotide Array: Eukaryotic Target Preparation and One-
color detection
Detection of mRNA
