Russian Journal of Bioorganic Chemistry

, Volume 33, Issue 2, pp 243–250 | Cite as

Study of the quality of aminated substrates used for hybridization analysis

  • E. A. Shlyapnikova
  • Yu. M. Shlyapnikov
  • V. N. Afanas’ev
  • G. V. Afanas’eva
  • A. V. Gavryushkin
  • I. P. Beletskii
Article

Abstract

A set of methods for analysis of the quality of aminated substrates that could be a basis for the large-scale manufacturing of biological microchips is suggested. The analysis includes the determination of the number of amino groups, their availability for the immobilization of phosphorylated oligonucleotides, and the characterization of surface properties of the substrates in respect to the nonspecific sorption of reagents during hybridization. A simple procedure was suggested for determination of the density/number of amino groups. It is based on the use of dimethoxytrityl chloride with the subsequent spectrophotometric determination of dimethoxytrityl cation. The availability of amino groups was estimated by covalent attachment of an oligonucleotide probe containing a fluorescently labeled group to the aminated surface and the subsequent comparison of the intensity of fluorescing zones formed on the chip. The sorption properties of the surface were investigated with the help of a model hybridization reaction. A comparative analysis of aminated glasses manufactured by various firms and in our laboratory showed that the glasses with the amino group density from 0.7 to 2.0 groups/nm2 prepared by our procedure have the best properties for the hybridization analysis.

Key words

DNA chips hybridization analysis oligonucleotide immobilization 

Abbreviations

APTES

3-aminopropyltriethoxysilane

BSA

bovine serum albumin

DAB

dimethylaminobenzidine

DMAP

4-dimethylaminopyridine

DMTrCl

dimethoxytrityl chloride

EDC

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

FAM

6-carboxyfluorescein

1xSSC

150 mM NaCl, 25 mM sodium citrate, pH 7.4

TNBS

2,4,6-trinitrobenzenesulfonate

SPDP

N-succinimidyl 3-(2-pyridyldithio)propionate

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Copyright information

© Pleiades Publishing, Inc. 2007

Authors and Affiliations

  • E. A. Shlyapnikova
    • 1
  • Yu. M. Shlyapnikov
    • 1
  • V. N. Afanas’ev
    • 1
  • G. V. Afanas’eva
    • 1
  • A. V. Gavryushkin
    • 1
  • I. P. Beletskii
    • 1
  1. 1.Institute of Theoretical and Experimental BiophysicsRussian Academy of SciencesPushchino, Moscow oblastRussia

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