Electrostatic Printing of Composite Features for Highly Uniform DNA Microarrays and Other Innovations in Microarray Technology P. Dextras, K. Guggenheimer, J. Thompson and A. Marziali Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada 3.2. Composite feature printing methodology 1. GenomeBC Technology Development Team • GenomeBC is a non-profit organization funded by GenomeCanada to fund and promote Genomics research in British Columbia. The Technology Development Team was established at UBC to provide shared technology development and engineering support for GenomeBC projects. • Our current focus is to investigate sources of technical error in microarray methodologies and to develop novel microarraying technologies. The two main projects being pursued are: The prototype is currently used to print a 50 M solution of a 25-mer oligonucleotide with a 5’ 6-FAM fluorescent tag. The current aim is to determine the optimum voltage, pulse width, spot spacing and time between prints for maximum uniformity. Fluorescence uniformity is measured by imaging the slide on an Applied Precision arrayWoRx scanner with a resolution of 4 m. The physical morphology is determined using a scanning electron microscope. iv. v. vi. A reverse voltage is applied across the capillary to flush out any unbound target. The reverse potential is kept low enough to prevent dissociating the hybridized DNA molecules. After removing the unbound target molecules the end of the capillaries are imaged with a laser confocal scanner. After scanning fresh LPA is pumped into the end of the capillary and another assay can be performed. Tagged target DNA 1. Electrostatic Printing of Composite Features for Highly Uniform DNA Microarrays Using a pulsed field droplet spotter, we are developing a new method for printing highly uniform microarray features. Replaceable Gel-based Hybridization Assay This device performs DNA hybridization experiments inside glass capillaries with both probe and target in free solution. • Square features are currently printed in order to simplify the LabView control software; however, any arbitrary shape could be printed. Originally, features were built up by printing in a raster pattern, but this resulted in mass transfer of solution towards the leading edge of the feature. A higher uniformity can be achieved by first printing the odd rows of the feature and then filling in the even rows. LPA bound oligo Capillary 2. • A B C D For information on other current technology development projects, refer to: www.physics.ubc.ca/~andre/ (A) Schematic of an oligo bound to an LPA molecule. (B) End of a capillary exposed to target solution (C) Electrokinetic injection of target molecules. (D) Reverse voltage applied to remove unbound target. 2. Microarray Feature Uniformity • Printing with contact devices such as tungsten pins often results in concentration and morphology variation. Fluorescence can be quenched or change emission wavelength based on the local concentration of dyes. Red/ Green ratios may vary from pixel to pixel (Brown, C. Goodwin, P. Sorger, P. 2001. “Image metrics in the statistical analysis of DNA microarray data.” PNAS 98 (16): 8944-8949.) Our goal is to develop a method of printing features which reduces non-uniformity in microarray data. Fluorescence image (above) and 3D plot (left) showing morphology variation in pinprinted microarray features. Scanning electron microscope image (above left), and array scanner fluorescence image in 2D (center) and 3D (right). 4.3 Current Progress A version of this device with a single capillary and a two colour laser confocal scanning system capable of imaging cy3 and cy5 has been built. Initial single colour tests with 6-FAM tagged oligos have shown that we can distinguish between an oligo perfectly matched to the probe and one with a complete mismatch (see below). Capillary • Pressure In Sample Reservoir • 3.3 • • Future direction of the project +/-/+ Objective • We plan to apply a conventional surface chemistry to the ITO-coated slides to covalently bond probe molecules for hybridization experiments. Multiple independently-addressable capillaries will be integrated into a single print head for high-speed printing of large microarrays. Laser LPA Reservoir High Voltage Supply PMT 3. Electrostatic Printing of Composite Features for Highly Uniform DNA Microarrays 3.1 Pulsed field droplet spotter 4.4 Fluorescence signal from a complementary oligo probe (left-top) and a non-complementary oligo probe (leftbottom). A schematic of a single capillary version of this Advantages over slide based arrays device (right) •Printing: the printing step has been reduced to simply pumping fresh LPA into the end of a capillary 4. Replaceable Gel-based Hybridization Assay • A non-contact printing device that is able to deliver small liquid volumes onto conductive substrates (Yogi, et. al. 2001. “On Demand Droplet Spotter for Preparing Pico- to-Femtoliter Droplets on Surfaces.” Anal. Chem. 73 (8): 1896-1902). Prints electrostatically when a voltage pulse is applied between the solution in the capillary and the substrate. A clear, electrically conductive printing surface is required (currently using an indium tin oxide coating on glass). The prototype system can print spots as small as 2µm in diameter. This makes it possible to construct large (~100µm) features from overlapping spots resulting in a high overall uniformity. 4.1 Device Overview We are currently developing a device for performing gel-based hybridization assays, with replaceable linear polyacrylamide (LPA) inside of a glass capillary. The goal of this work is to develop a device for performing extremely high throughput hybridization assays on a relatively small number (hundreds) of SNP’s or genes. •Imaging: imaging is simplified because the location of the ‘spots’ is always fixed, and the morphology of the spot is always the same. •Hybridization Speed: because target is electrokinetically injected into the capillary you don’t have to wait for targets to diffuse to spots, also steric hindrance is less of a problem. 4.5 Future Work • •Optimize the single capillary device for distinguishing single base pair mismatches. 4.2 Device Operation i. ii. iii. • The first step in the hybridization assay is to fill a capillary with LPA in which acrylamide modified probe oligonucleotides have been incorporated into the polymer chains Next the end of the capillary is exposed to a solution of tagged target DNA. A voltage is then applied across the capillary to electrokinetically inject target DNA into the capillary, here complementary target DNA will hybridize to the bound probe molecules. •Expand to a multiple capillary device, with the ultimate goal of incorporating 96 or 384 capillaries in a single device. Here capillaries would be close packed at the hybridization end, and the other end would be rearrayed into microtiter plate format, where each well in a microtiter plate would contain a different probe oligonucleotide. Acknowledgements We thank GenomeBC for financial support. We also thank Scott Tebbutt and the iCAPTUR 4E Center, Colleen Nelson, Jeff Zeznik and the Jack Bell Research Centre for their contributions.