An Inexpensive and Simple Method for Thermally Stable Immobilization of DNA on an Unmodified Glass Surface: UV Linking of Poly(T)10-poly(C)10–Tagged DNA Probes

Abstract

Microarrays printed on glass slides are often constructed by covalently linking modified oligonucleotide probes to a derivatized surface at considerable expense. In this article, we demonstrate that 14-base oligonucleotides with a poly(T)10 - poly(C)10 tail (TC tag), but otherwise unmodified, can be linked by UV light irradiation onto a plain, unmodified glass surface. Probes immobilized onto unmodified glass microscope slides performed similarly to probes bound to commercial amino-silane-coated slides and had comparable detection limits. The TC-tagged probes linked to unmodified glass did not show any significant decrease in hybridization performance after a 20 min incubation in water at 100 degrees C prior to rehybridization, indicating a covalent bond between the TC tag and unmodified glass. The probes were used in thermal minisequencing cycling reactions. Furthermore, the TC tag improved the hybridization performance of the immobilized probes on the amino-silane surface, indicating a general benefit of adding a TC tag to DNA probes. In conclusion, our results show that using TC-tagged DNA probes immobilized on an unmodified glass surface is a robust, heat-stable, very simple, and inexpensive method for manufacturing DNA microarrays.

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…Work reported by the Wolff group has demonstrated an approach to the covalent immobilization of specific primers tagged at the 5' termini with a homopolymer sequence polyT 10 -polyC 10 (TC tag) on unmodified glass slides using simple UV cross-linking [17][18][19]. This resulted in a more stable covalent attachment to the glass surface which sometimes can be an issue in solid-phase amplification [18,20].…”

“…This resulted in a more stable covalent attachment to the glass surface which sometimes can be an issue in solid-phase amplification [18,20].…”

“…2 (A-D)). The addition to the solution of a small amount, usually less than 10%, of the same primer as immobilized on the solid-support allows for increased efficacy of the amplification [3,13,17,18]. Initially, the reaction compartment comprises of solid-phase primers as well as forward (fwd) and reverse (rev) primers in an asymmetric ratio (A); at the start of the reaction the PCR proceeds preferably in the liquid phase, until the forward primer is depleted (B); then the solid-phase PCR dominates where the immobilized primer is extended by DNA polymerase (C) and (D).…”

Selective binding of oligonucleotide on TiO 2 surfaces modified by swift heavy ion beam lithography

“…UV light exposure is frequently use for sterilization of surfaces and it has also been employed satisfactorily for photo-immobilization [17]. UV light can also promote chemical changes of the exposed materials.…”

Microfluidic DNA microarrays in PMMA chips: streamlined fabrication via simultaneous DNA immobilization and bonding activation by brief UV exposure

“…UV exposure has also been used to directly attach oligonucleotides to surfaces including unmodified PMMA. Dufva et al and Gunadson et al have demonstrated the use of poly(T)10-poly(C)10-tagged DNA probes to unmodified and agarose-coated glass surfaces as well as PMMA (Dufva et al 2006;Gudnason et al 2008) and Kimura used oligonucleodtides incorporating a 5'-end poly(dT) tail and and an undisclosed spacer group (Kimura 2006) for attachment to unmodified PMMA.…”