AVS 47th International Symposium
    Biomaterial Interfaces Thursday Sessions
       Session BI+NS-ThA

Invited Paper BI+NS-ThA1
Substrate and Attachment Chemistry Effects on Adsorption and Single-Base Mismatch Discrimination on Immobilized Oligonucleotide Arrays

Thursday, October 5, 2000, 2:00 pm, Room 202

Session: Biosensors
Presenter: J.E. Forman, Zyomyx, Inc.
Authors: J.E. Forman, Zyomyx, Inc.
L. Gamble, Zyomyx, Inc.
R.S. Gascon, Zyomyx, Inc.
J.I. Henderson, Zyomyx, Inc.
A.D. Suseno, Zyomyx, Inc.
P. Wagner, Zyomyx, Inc.
Correspondent: Click to Email
Hybridization efficiency and ability to discriminate between perfect match and single-base mismatch target sequences are fundamental to performance of arrays of covalently immobilized nucleic acids (probes). Two factors that significantly contribute to array performance are density and orientation of immobilized probes. Probe density not only controls how much bound target is adsorbed, but also affects the kinetics and thermodynamics of duplex formation. Perturbations to duplex formation occur when the spacing of probes is close enough to force inter-probe association and crowding, phenomena that interfere with target sequence adsorption. However, these perturbations can induce beneficial effects in array performance. For example, crowded nucleic acid surfaces demonstrate lower apparent melting temperatures (T@sub m@'s) than the solution phase, but also show discrimination between matched and mismatched sequences under low stringency conditions. The orientation of the probe is highly dependent on the method of immobilization to the surface. Non-optimal attachment can orient the probe in a way that interferes with duplex formation, or such that it becomes buried within the surface and inaccessible to the target sequence. The substrate used for array preparation ultimately controls both density and orientation; optimization of the substrate can enhance array performance in an assay. Especially interesting is a precise control of probe density, where the density effects alter the T@sub m@'s of immobilized sequences to allow a broad range of sequence to be analyzed in a single temperature assay. We have been exploring a variety of silane modified substrates for the preparation of oligonucleotide arrays, focusing on immobilization of the probe through amine or thiol moieties. The effect of density and attachment chemistry on target adsorption and single-base mismatch discrimination with single and double stranded oligonucleotide target sequences will be presented.