The manufacturer protocol used for the Nimblegen human custom array was performed according to the experimental protocol described in: Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nat Biotechnol. 1999 Oct;17(10):974-8. Silanization of microscope slides. Microscope slides were prepared according to the method of McGall and colleagues8. Briefly, soda lime glass microscope slides (Fisher Scientific Co., Itasca, IL) were soaked in a bath of Nanostrip (Cyantek, Fremont, CA) for 15 min, water, 1% (vol/vol) aqueous HCl for 1 min, 70°C 10% (wt/vol) aqueous NaOH for 3 min, water, 1% (vol/vol) solution of N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide (Lancaster Synthesis, Windham, NH) for 15 min. The slides were then rinsed in 2-propanol for 5 min, water for 5 min, and then dried in a spin dryer for 5 min at 100°C under nitrogen. Slides were then stored desiccated at -20°C until use. Synthesis of photolabile phosphoramidites. The MeNPOC-HEG-cyanoethylphosphoramidite (CEP) and MeNPOC-base-CEPs were synthesized according to the protocol described by McGall and colleagues8. Briefly, 3,4-(methylenedioxy)acetophenone (Aldrich Chemical Co., Milwaukee, WI) was reacted with glacial acetic acid and 70% (vol/vol) HNO3 to produce methyl 3,4-(methylenedioxy)-6-nitrophenyl ketone. This molecule was reacted with sodium borohydride to produce (R,S)-1-(3,4-(methylenedioxy)-6-nitrophenyl)ethanol, which was then reacted with phosgene to produce (R,S)-1-(3,4-(methylenedioxy)-6-nitrophenyl)ethyl chloroformate (MeNPOC-Cl). This molecule was attached to hexaethylglycol and A(PAC), C(ibu), and G(ibu). These molecules were then converted to phosphoramidites by reacting with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite and diisopropyl ammonium tetrazolide to produce the final products. Coating of silanized microscope slides with MeNPOC-HEG. Silanized microscope slides were attached to the reaction chamber and MeNPOC-HEG-CEP (50 mM) was delivered to the chamber via a standard DNA synthesizer (ABI 392), which was programmed to deliver reagents for DNA synthesis. Patterned photodeprotection of MeNPOC. The MAS was used to deliver the desired photon using control software (Modularis, Chicago, IL) that designates the desired exposure time and displays the appropriate virtual mask at the correct time and place. The intensities given above for various experiments were measured at the image plane for an all-white image using an International Light UV Dosimeter (Newburyport, MA) with a calibrated detector for 365 nm radiation. A typical intensity was 19.5 mW/cm2 for a lamp with 227 h of prior use. (Intensity increased to 28.5 mW/cm2 with a new Hg arc lamp). Staining of deprotected hydroxyl groups with FluorePrime fluorescein amidite. A mixture of FluorePrime (Amersham Pharmacia Biotech, Uppsala, Sweden) and DMT-Thymidine-CEP (Glen Research, Sterling, VA) 1:10 was diluted with anhydrous acetonitrile to a final concentration of 50 mM amidites. This mixture was delivered to the chamber via the DNA synthesizer during a cycle of standard phosphoramidite chemistry. After coupling of the Fluoreprime to the microscope slide, the slide was removed from the chamber. The slide was soaked for 15 min in a solution of ethylenediamine−EtOH (1:1) to remove protecting groups on the phosphate and fluorescein molecules and then rinsed for 5 min in water and spun dry. Visualization of patterned fluorescein-labeled slides. The slides were viewed with a Bio-Rad (Bio-Rad Laboratories, Hercules, CA) MRC-1024 laser scanning confocal microscope. The 488 nm line of an Ar/Kr laser was the excitation source, and a 530 nm bandpass filter was used in front of a photomultiplier tube to collect the emission from the fluorescein. Images were acquired using both direct-mode and time-domain Kalman filtering of five image frames. Oligonucleotide array fabrication and hybridization. The MAS was used to pattern MeNPOC-phosphoramidites in dioxane. The oligonucleotide 5'-AGGTCATTACAGCGAGAG-3' was selected from the Arabidopsis CPK6 gene using the Primer3 program10 developed at Massachusetts Institute of Technology, Cambridge, MA). Three additional 18-mers were created by changing one, two, or three bases of the original sequence. After synthesis of the oligonucleotide probes, the slide was soaked in a solution of ethylenediamine−ethanol (1:1) for 2 h to remove the protecting group and then washed with water. The slide was hybridized with 200 l of 100 nM synthetic biotin-labeled oligonucleotide target 5'-biotinCTCTCGCTGTAATGACCT-3' (Genosys Biotechnologies, The Woodlands, TX). The hybridization solution contained 100 mM (2-[N-Morpholino]ethanesulphonic acid (MES), 1 M NaCl, 20 mM EDTA, and 0.01% (vol/vol) Tween 20. The hybridization was carried out overnight at room temperature in a hybridization chamber consisting of the DNA chip slide, a Kalrez (DuPont Dow Elastomers, Newark, DE) gasket, and a glass coverslip. After hybridization was complete, the array wash washed with a nonstringent wash buffer (6 SSPE [0.9M NaCl, 60 mM HaH 2PO4, 6 mM EDTA (pH7.4)]) and 0.01% [vol/vol] Tween 20) and stained with streptavidin phycoerythrin (10 g/ml) in 100 mM MES, 1 M Na +, and 0.05% Tween 20 for 30 min. The array was then washed with the nonstringent buffer and imaged with a Bio-Rad confocal microscope.
