Derivatization of Glass and Polypropylene Surfaces

Markus Beier1, Jörg D. Hoheisel2

1 Febit AG, Mannheim, 2 Deutsches Krebsforschungszentrum, Heidelberg
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 12.4
DOI:  10.1002/0471142700.nc1204s18
Online Posting Date:  October, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes the derivatization of solid support media, particularly glass and polypropylene, with a linker system by an iterative process that forms a dendrimeric structure, thereby increasing stepwise the loading capacity of the surface. The procedure permits the production of various types of linkers whose characteristics can be tailored to the requirements of the eventual applications. The specific modifications described here are used either for attaching prefabricated DNA oligonucleotides, PCR products, and peptide nucleic acid (PNA) oligomers, or for in situ synthesis of DNA microarrays.

Keywords: solid support; etching; silanization; linker; immobilization of biomolecules; microarray

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Derivatization of Solid Support
  • Support Protocol 1: Silanization of Glass Slides
  • Support Protocol 2: Quality Control of Derivation Reactions
  • Support Protocol 3: Immobilization of 5′‐Amino‐Labeled Nucleic Acid Compounds
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Derivatization of Solid Support

  Materials
  • Acetone
  • Nitrogen
  • Diisopropylethylamine (DIPEA)
  • Acetonitrile, anhydrous
  • 4‐Nitrophenyl chloroformate or acryloyl chloride
  • Dichloroethane, dry
  • Amine compound—e.g., tetraethylenepentamine; 1,4‐bis‐(3‐aminopropoxy)butane; 4‐aminomethyl‐1,8,‐octadiamine; 4,7,10‐trioxa‐1,13‐tridecandiamine; N,N‐dimethyl‐1,6‐hexadiamine; 2‐(2‐aminoethoxy)ethanol; jeffamine; 3‐amino‐1,2‐propandiol
  • Dimethylformamide (DMF), dry and amine free
  • Methanol, reagent grade
  • 18 × 8–cm polypropylene vessels with tight‐fitting lids
  • Solid support medium (e.g., glass, polypropylene foil, nylon membrane, polypropylene membrane) with anchoring groups in place on surface (e.g., see protocol 2 for silanizing glass)
  • Orbital shaker

Support Protocol 1: Silanization of Glass Slides

  Materials
  • 10% (w/v) aqueous NaOH
  • 1% (v/v) aqueous HCl
  • Methanol, reagent grade
  • 3% (w/v) aminopropyltrimethoxysilane in 95% (v/v) methanol
  • Nitrogen
  • Glass microscope slides (e.g., Menzel‐Gläser, Germany)
  • Several glass vessels with lids (e.g., hematology staining jar)
  • Orbital shaker
  • Polypropylene vessel with lid
  • Bath sonicator
  • 110°C oven

Support Protocol 2: Quality Control of Derivation Reactions

  • Control strip: hydroxylated or aminated solid support (see protocol 1)
  • Amine‐free DMF containing 0.05% bromphenol blue
  • Ethanol, reagent grade
  • 20% piperidine in DMF
  • Spectrophotometer, 605 nm

Support Protocol 3: Immobilization of 5′‐Amino‐Labeled Nucleic Acid Compounds

  • Phenylene diisothiocyanate (PDITC) or dimethylsuberimidate dihydrochloride (DMS)
  • 10% (v/v) anhydrous pyridine in amine‐free DMF (for PDITC)
  • Saturated aqueous sodium bicarbonate (NaHCO 3; for DMS)
  • 5′‐Amino‐labeled nucleic acid
  • Diisopropylethylamine (DIPEA)
  • 1 mM Tris⋅Cl, pH 7.5 (see appendix 2A; optional)
  • 6‐Amino‐1‐hexanol
  • Amino‐functionalized glass slides or polypropylene sheets (see protocol 1)
  • 37°C humid chamber
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   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 of quantitative photolithographic synthesis of individually quality checked DNA microarrays. Nucl. Acids Res. 28:e11.
   Diehl, F., Grahlmann, S., Beier, M., and Hoheisel, J.D. 2001. Manufacturing DNA‐microarrays of high spot homogeneity and reduced background signal. Nucl. Acids Res. 29:e38.
   Pease, A.C., Solas, D., Sullivan, E.J., Cronin, M.T., Holmes, C.P., and Fodor, S.P.A. 1994. Light‐generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. U.S.A. 91:5022‐5026.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library