Novel Reagents and Procedures for Immobilization of DNA on Glass Microchips for Primer Extension

Abstract

A new method for attaching oligodeoxyribonucleotides to glass involving monoalkoxylated and dialkoxylated silanes and bromoacetamide/phosphorothioate linking chemistry has been developed. Three novel bromoacetamide silanes were synthesized for derivatization of glass microscope slides by traditional dipping methods. A thin film method that conserves silane and provides a consistent protocol for test experiments was also used. Oligonucleotides bearing 5′-phosphorothioates were synthesized by literature methods. Immobilization conditions were initially established by treatment of bromoacetamidosilyl slides with fluoresceinated oligonucleotides, which were imaged by confocal fluorescence microscopy. Spotting can be accomplished in water at oligonucleotide concentrations down to 0.1 mM. Oligonucleotides immobilized using this method can serve as primers for templated, polymerase-based extension reactions with a fluoresceinated dideoxynucleotide terminator. When such primers are formatted into small arrays, specific extension is observed only in the presence of complementary template, with the amount of immobilized primer reflected in the fluorescence signal.

Engineering Silicon Oxide Surfaces Using Self‐Assembled Monolayers

“…[194] Other reported strategies to attach biological molecules to SiO 2 surfaces involve the reactions of sulfanyl-terminated SAMs with disulfide-modified oligonucleotides or heme proteins, [195,196] iodopropyl monolayers with sulfanyl-containing proteins, [197] alcohols with a silyl chloride surface, [198] and bromoacetamide SAMs with phosphorothioate-functionalized oligonucleotides. [199] 4. Engineering the Silicon Oxide Surface Using Self-Assembled Monolayers…”

Gestaltung der Siliciumoxidoberfläche durch selbstorganisierte Monoschichten

“…[194] Weitere Methoden zur Verknüpfung biologischer Moleküle mit SiO 2 -Oberflächen nutzen Reaktionen von sulfanylterminierten SAMs mit disulfidmodifizierten Oligonucleotiden oder Hämoproteinen, [195,196] Iodpropyl-Monoschichten mit Sulfanylgruppen enthaltenden Proteinen, [197] Alkoholen mit einer Silylchlorid-Oberfläche [198] und BromacetamidSAMs mit Phosphorothioat-funktionalisierten Oligonucleotiden. [199] 4. Gestaltung von Siliciumoxid-Oberflächen durch selbstorganisierte Monoschichten…”

Silicon Based Nanocoatings on Metal Alloys and Their Role in Surface Engineering

“…This also led to the determination of the binding constants between an immobilized tripeptide and vancomycin using confocal fluorescence detection [321]. Some of the other strategies for the attachment of biological molecules to silicon oxide surfaces are reactions of sulfanyl terminated SAMs with disulphide modified oligonucleotides or heme proteins [322,323], iodopropyl monolayers with sulfanyl containing proteins [324], alcohols having a silyl chloride surface [325] and bromoacetamide SAMs with phosphorothiolate functionalized oligonucleuotides [326]. Arya et al have developed a cholesterol biosensor by covalently immobilizing cholesterol oxidase onto an amino terminated alkoxy silane deposited on indium tin oxide (ITO) electrodes [327].…”