DNA Microarray Preparation by Light‐Controlled In Situ Synthesis

Markus Beier1, Jörg D. Hoheisel1

1 Deutsches Krebsforschungszentrum, Heidelberg
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 12.5
DOI:  10.1002/0471142700.nc1205s20
Online Posting Date:  April, 2005
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This unit describes the in situ synthesis of DNA microarrays using a light‐controlled process. In a highly parallel fashion, multiple oligonucleotide probes are created from scratch on a glass support using phosphoramidite building blocks carrying a photolabile protecting group. Spatial resolution is achieved by carefully controlling the illumination of defined spots on the glass substrate, on which the oligonucleotide probes are synthesized using straightforward phosphoramidite chemistry.

Keywords: oligonucleotide microarrays; genomics; biochip; photolithography; DNA synthesizer; photolabile; NPPOC

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Assembly of an Apparatus for Light‐Directed Synthesis of Oligonucleotide Microarrays
  • Basic Protocol 2: Light‐Directed in Situ Synthesis of Oligonucleotide Microarrays
  • Basic Protocol 3: Hybridization to Oligonucleotide Microarrays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Assembly of an Apparatus for Light‐Directed Synthesis of Oligonucleotide Microarrays

  • 50 mM iodine in 7:1:2 (v/v/v) acetonitrile/pyridine/water
  • Flow cell apparatus (custom‐made; Fig. ), including the following:
    • Teflon reactor
    • Aluminum base plate
    • Rectangular rubber seal
    • Nonderivatized microscope slide (for optimization of DNA synthesizer program)
    • Derivatized microscope slide (for synthesis of microarray; see unit 12.4)
    • Stainless steel top plate with inner mask (16 to 64 holes)
    • DNA synthesizer (e.g., Eppendorf D200)
  • 100‐watt mercury lamp (e.g., Leica)
  • 365‐nm interference filter (e.g., Owis)
  • Optical shutter (e.g., Owis)
  • Optical bench (e.g., Owis) and assorted optical hardware

Basic Protocol 2: Light‐Directed in Situ Synthesis of Oligonucleotide Microarrays

  • Standard DNA synthesizer reagents (Proligo):
    • Activator (e.g., pyridine hydrochloride, dicyanoimidazole, tetrazole)
    • Anhydrous acetonitrile (for wash steps)
    • Oxidizing reagent
    • Capping reagents
  • Irradiation buffer: 50 mM diisopropylethylamine (DIPEA) in acetonitrile
  • NPPOC‐protected phosphoramidite solutions in acetonitrile (dA, dC, dG, and T; concentration, 0.1 M for each; see unit 12.3)
  • 25% (v/v) ammonia in water
  • Nitrogen stream
  • Assembly for light‐directed oligonucleotide microarray synthesis (see protocol 1)
  • Removable black tape (e.g., standard laboratory tape): one precut piece of sufficient size to cover the entire stainless steel mask, plus precut pieces to cover each individual hole in the mask
  • Polypropylene jar with lid
  • Orbital shaker
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C)
NOTE: Immediately before being used in this protocol, phosphoramidites (prepared as in unit 12.3) should be dried and dissolved in acetonitrile. Care should be taken to prevent phosphoramidite solutions from absorbing humidity from the atmosphere.

Basic Protocol 3: Hybridization to Oligonucleotide Microarrays

  • Target oligonucleotide sample
  • SSARC hybridization buffer (see recipe)
  • Microscope slide containing oligonucleotide microarray (see protocol 2)
  • Nitrogen stream
  • Coverslip
  • Polypropylene vessel with lid
  • Fluorescence scanner, preferably laser or charge‐coupled device (CCD), and image analysis software
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Literature Cited

Literature Cited
   Baum, M., Bielau, S., Rittner, N., Schmid, K., Eggelbusch, K., Dahms, M., Schlauersbach, A., Tahedl, H., Beier, M., Güimil, R., Scheffler, M., Hermann, C., Funk, J.M., Wixmerten, A., Rebscher, H., Hönig, M., Andreae, C., Büchner, D., Moschel, E., Glathe, A., Jäger, E., Thom, M., Greil, A., Bestvater, F., Obermeier, F., Burgmaier, J., Thome, K., Weichert, S., Hein, S., Binnewies, T., Foitzik, V., Müller, M., Stähler, C.F., and Stähler, P.F. 2003. Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling. Nucl. Acids Res. 31:e151.
   Beier, M. and Hoheisel, J.D. 1999. Versatile derivatisation of solid support media for covalent bonding on DNA‐microchips. Nucl. Acids Res. 27:1970‐1977.
   Beier, M. and Hoheisel, J.D. 2000. Production by quantitative photolithographic synthesis of individually quality checked DNA microarrays. Nucl. Acids Res. 28:e11.
   Beier, M., Stephan, A., and Hoheisel, J.D. 2001. Synthesis of photolabile 5′‐O‐phosphoramidites for the production of microarrays of inversely oriented oligonucleotides. Helv. Chim. Acta 84:2089‐2095.
   Bühler, S., Lagoja, I., Giegrich, H., Stengele, K.P., and Pfleiderer, W. 2004. New types of very efficient photolabile protecting groups based upon the [2‐(2‐nitrophenyl)propoxy]carbonyl (NPPOC) moiety. Helv. Chim. Acta 87:620‐659.
   Fodor, S.P., Read, J.L., Pirrung, M.C., Stryer, A., Liu, A., and Solas, D. 1991. Light‐directed, spatially addressable parallel chemical synthesis. Science 251:767‐773.
   Fodor, S.P., Rava, R.P., Huang, X.C., Pease, A.C., Holmes, C.P., and Adams, C.L. 1993. Multiplexed biochemical assays with biological chips. Nature 364:555‐556.
   Gao, X., Yu, P., LeProust, E., Sinigo, L., Pellios, J.P., and Zang, H. 1998. Oligonucleotide synthesis using solution photogenerated acids. J. Am. Chem. Soc. 120:12698‐12699.
   Giegrich, H., Eisele‐Bühler, S., Hermann, C., Kvasyuk, E., Charubala, R., and Pfleiderer, W. 1998. New photolabile protecting groups in nucleoside and nucleotide chemistry—synthesis, cleavage mechanisms and applications. Nucleosides Nucleotides 17:1987‐1996.
   Hasan, A., Stengele, K.P., Giegrich, H., Cornwell, P., Isham, K.I., Sachleben, R., Pfleiderer, W., and Foote, R.S. 1997. Photolabile protecting groups for nucleosides: Synthesis and photodeprotection rates. Tetrahedron 53:4247‐4264.
   McGall, G.H., Barone, A.D., Diggelman, M., Fodor, S.P., Gentalen, E., and Ngo, N. 1997. The efficiency of light‐directed synthesis of DNA arrays on glass substrates. J. Org. Chem. 119:5081‐5090.
   Pease, A.C., Solas, D., Sullivan, E.J., Cronin, M.T., Holmes, C.P., and Fodor, S.P. 1994. Light‐generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. U.S.A. 91:5022‐5026.
   Pirrung, M.C. and Bradley, J.C. 1995. Comparison of methods for photochemical phosphoramidite‐based DNA synthesis. J. Org. Chem. 60:6270‐6276.
   Pirrung, M.C., Fallon, L., and McGall, G. 1998. Proofing of photolithographic DNA synthesis with 3′,5′‐dimethoxybenzoinoxycarbonyl‐protected deoxynucleoside phosphoramidites. J. Org. Chem. 63:241‐246.
   Singh‐Gasson, S., Green, R.D., Yue, Y.J., Nelson, C., Blattner, F., Sussman, M.R., and Cerrina, F. 1999. Maskless fabrication of light‐directed oligonucleotide microarrays using a digital micromirror array. Nat. Biotechnol. 17:974‐978.
   Southern, E.M., Case‐Green, S.C., Elder, J.K., Johnson, M., Mir, K., Wang, L., and Williams, J.C. 1994. Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids. Nucl. Acids Res. 22:1368‐1373.
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