DNA Microarrays
What is a microarray A surface on which sequences from thousands of different genes are covalently attached to fixed locations (prob...
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 %
Phosphoramidite chemistry at 98% 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
C
O
O O CH3
BASE
O
C CH CH2 C NH CH CH CH S O 2 2 2 2 i O O 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
Protection of Base Amino Groups O O NH C
HN
N C O
N
N
CH CH3 CH3
N
N
N
NH
O O CH 3
HN O
NH C
N
N O
N
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’
Natural DNA 5’
3’
CPG
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
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)
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.
Gridding: identify spots (automatic, semiautomatic, manual) Segmentation: separate spots from background. Fixed circle (B), Adaptive circle C, Adaptive shape (D), Histogram Intensity extraction: mean or median of pixels in spot 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 Onecolor detection