Immobilization and hybridization of DNA in a sugar polyacrylate hydrogel.
Using a non-contact microarrayer, amine-terminated probe oligonucleotides representing 20-, 50-, and 70-mer fragments of the fliC gene were covalently coupled into three-dimensional regions in a "sugar polyacrylate" hydrogel based on poly(6-acryloyl-beta-O-methyl galactopyranoside-co-aminopropyl methacrylamide). The arrayer deposited the solution containing ssDNA probes in discrete regions on the surface of the gel (i.e. as a droplet with a ca. 450 microm diameter), allowing penetration and attachment of the ss DNA within the three dimensional region of the gel. The attachment was mediated by the homobifunctional crosslinker bis-succinimidyl suberate. Confocal microscopy showed the density of attached probe DNA was greatest in the interior-most regions of the gel volume. Target ssDNA (20- and 70-mer) was able to diffuse through the gel and undergo successful hybridization with the probes. For target ssDNA in the concentration range 0.19 microM to 6.0 microM, there was a linear correlation between DNA concentration and the fluorescence of the gel region where hybridization occurred.
Soto
Carissa M
CM
Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, 4555 Overlook Ave. SW, Washington, DC 20375, USA. cmsoto@cbmse.nrl.navy.mil
Patterson
Charles H
CH
Charles
Paul T
PT
Martin
Brett D
BD
Spector
Mark S
MS
D000180:Acrylic Resins / Q000737:chemistry*; D005690:Galactose / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
D000180:Acrylic Resins; D020100:Hydrogels; D009838:Oligodeoxyribonucleotides; C006903:carbopol 940; D005690:Galactose