Analytical Biochemistry 2006-10-15

N-(3-Triethoxysilylpropyl)-6-(N-maleimido)-hexanamide: An efficient heterobifunctional reagent for the construction of oligonucleotide microarrays.

Jyoti Choithani, P Kumar, K C Gupta

Index: Anal. Biochem. 357(2) , 240-8, (2006)

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Abstract

Synthesis of a new heterobifunctional reagent, N-(3-triethoxysilylpropyl)-6-(N-maleimido)-hexanamide (TPMH), for the preparation of oligonucleotide microarrays is described. Its triethoxysilyl function is specific toward virgin glass surface and maleimide function undergoes conjugate addition to 3'- or 5'-thiol-modified oligonucleotides. The construction of microarrays using TPMH has been realized via two routes. In Route A, TPMH was reacted first with 3'- or 5'-thiol-modified oligonucleotide under microwaves, thereby producing oligonucleotide-triethoxysilyl conjugate in 15min, which was then brought in contact with virgin glass microslide, resulting in immobilization of an oligonucleotide sequence. In Route B, immobilization involves generation of maleimide functions on virgin glass surface by the reaction with TPMH, followed by coupling with thiol-modified oligonucleotides under microwaves in 15 min to produce surface-bound oligonucleotides. The microarrays constructed using both routes were analyzed by hybridizing with tetrachlorofluorescein-labeled complementary oligonucleotide. Subsequently, these microarrays were successfully used in the discrimination of single and double nucleotide mismatches based on fluorescence intensity.