A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes.
N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.
Urdea
M S
MS
Chiron Corporation, Emeryville, CA 94608.
Warner
B D
BD
Running
J A
JA
Stempien
M
M
Clyne
J
J
Horn
T
T
D000469:Alkaline Phosphatase / Q000032:analysis; D003124:Colorimetry; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000032:analysis; D006735:Horseradish Peroxidase / Q000032:analysis; D008163:Luminescent Measurements; D008165:Luminol / Q000031:analogs & derivatives / Q000032:analysis; D008722:Methods; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000138:chemical synthesis; D009943:Organophosphorus Compounds / Q000138:chemical synthesis
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D003841:Deoxycytidine; C056441:N(4)-(N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl)-5'-O-dimethoxytrityl-5-methyl-2'-deoxycytidine-3'-N,N-diisopropylmethylphosphoramidite; C032487:isoluminol; D008165:Luminol; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase