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 Spotted Arrays DNA probes are synthesized
and then “spotted” onto the microarray
Customized for each experiment “in-lab”
Low cost Oligonucleotide
microarrays DNA oligonucleotides are
synthezised directly on the microarray
Commercial arrays (Affymetrix, Nimblegen and Combimatrix)
expensive
Detection Two-color detection Spotted Array: Compare two
samples by labeling with two different flurophores and analyzing on the same array Cy5 (red) and Cy3 (green)
One-color detection Determine gene expression
level One array per sample
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 %
50 %
0 %
10 bases 20 bases
Phosphoramidite chemistry at 98% coupling efficiency
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 2 O
CH 2 C O
NH CH 2 CH
2 CH 2 Si
O O C
O
O
BASE O C
O
CH 3
O
CH 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
Protection of Base Amino Groups
NH
N N
N O
HN C O CH
CH 3 CH 3
HN N
O
O
CH 3
N N
NH
O
C O
N N N
N NH C
O
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
CPG 3’5’
Oligonucleotide
Natural DNA 3’5’
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)
0.2 umole
1 umole
10 umole
20 - 25
5 - 10
100 - 120
800 - 1000
Analysis & Purification
PAGE
HPLC
CE (capillary electrophoresis)
OPC (oligonucleotide Purification Cartridge)
Quantitation & Storage
Storage: stable with little or no degradation for
long periods of time (over a year)
UV spectroscopy: Measuring OD at 260 nm
1 OD 260 for ssDNA = 33 ug/ml
Alternative chemistries
5' modified oligonucleotides
RNA oligoribonucleotides (using 2' silyl 5' DMT-CE phosphoramidite RNA monomers)
Antisense (phosphorothioate)
5' modified oligonucleotides
Aminolink
Enzyme - labeled
Biotin - labeled
Fluorescein-lebeled
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
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.
Types of Microarray Platforms
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
Allows a direct comparison between two different samples
Spotted Arrays: Sample Preparation and Two-color Detection
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
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