UV light-aided immobilization of oligonucleotides on glass surface using N -(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) and detection of single nucleotide mismatches

Nucleic Acids Research Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi Univer-

sity Campus, Delhi-110 007, India

E-mail : kcgupta@igib.res.in                         Fax : 91-11-27667471

Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110 007, India

E-mail: gandhirp@yahoo.com                          Fax : 91-11-27666248

Manuscript received 10 December 2003

A novel heterobifunctional reagent, N-(3-trinuoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) has been developed for construction of oligonucleotide microarrays. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one, (A), mercapto- or aminoalkyl-oligonucleotides react with NTPAC to form oligonucleotide-anthraquinone conjugate which, in a subsequent reaction with modified glass surface under UV light (365 nm), results in surface bound oligonucleotides. In the second route, (8), the reagent, NTPAC, is allowed to react first with modified glass surface under UV light whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with mercapto- or aminoalkyl oligonucleotides to generate polymer bound oligonucleotides. The constructed microarrays are successfully used in single nucleotide mismatch detection by hybridizing these with fluorescein labeled complementary oligonucleotides. The difference in fluorescence signal is used as an indicator of mismatch.