Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics.
Polyacrylamide supports, in a range of pore sizes, were investigated as nucleic acid affinity matrices for the detection of target DNA or RNA sequences using a sandwich hybridization format. Bromoacetyl and thiol oligonucleotide derivatives were covalently linked to sulfhydryl- and bromoacetyl-polyacrylamide supports with greater than 95% end-attachment efficiencies. These polyacrylamide-oligonucleotide supports were further derivatized with anionic residues to provide multi-functional supports which show low non-specific binding for non-complementary nucleic acids. While all the polyacrylamide-oligonucleotide supports capture complementary oligonucleotides with high affinity, the pore size was found to be a critical parameter in sandwich hybridization reactions. The superior hybridization characteristics of the Trisacryl support was ascribed to a combination of its macroporous nature, hydrophilicity and the terminal attachment of its capture oligonucleotides.
Fahy
E
E
Life Sciences Research Laboratory, Baxter Diagnostics Inc., San Diego, CA 92121.
Davis
G R
GR
DiMichele
L J
LJ
Ghosh
S S
SS
D000085:Acetates / Q000737:chemistry; D000180:Acrylic Resins / Q000737:chemistry*; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D012313:RNA / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D000099166:Polyacrylamides
D000085:Acetates; D000180:Acrylic Resins; D004247:DNA; D009841:Oligonucleotides; D012313:RNA; D013438:Sulfhydryl Compounds; D000099166:Polyacrylamides; C016274:bromoacetate