Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase.
Synthetic oligonucleotides were tailed at the 3' end using terminal deoxynucleotidyl transferase. Nucleotide triphosphates with free primary amines at the end of side chains were compared for their tailing efficiency and/or detection sensitivity, using biotin-11-dUTP as a reference. Free primary amines were tagged with activated biotin or fluorescein isothiocyanate. The probes were then detected with either streptavidin-alkaline phosphatase complex or anti-fluorescein antibodies and alkaline phosphatase-conjugated secondary antibodies. Tailing conditions were optimized and the probes were tested for detection of Escherichia coli ST1a enterotoxin DNA and rotavirus RNA.
Kumar
A
A
Station de Recherches de Virologie et d'Immunologie INRA, Thiverval-Grignon, France.
Tchen
P
P
Roullet
F
F
Cohen
J
J
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004253:DNA Nucleotidylexotransferase / Q000032:analysis*; D004254:DNA Nucleotidyltransferases / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D012401:Rotavirus / Q000032:analysis; D013950:Thymus Gland / Q000201:enzymology
D000345:Affinity Labels; D004269:DNA, Bacterial; D004768:Enterotoxins; D009841:Oligonucleotides; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase