Acetoxy-acetylaminofluorene-modified dGTP can be used to label oligonucleotides or DNA enzymatically.
A competitive enzyme immunoassay using a bispecific monoclonal antibody (Bi-MAb) was developed to quantify acetylaminofluorene (AAF) adducts fixed on DNA and then compared to the spectrophotometric method. It was shown that this simple method allowed the measurement of as low as 2 pmol per assay of AAF bound to DNA. This technique was used to monitor synthesis and purification of N-acetoxy-N-2-acetylaminofluorene modified dGTP (AAF-dGTP). It was shown that AAF-dGTP can be a substrate for the terminal deoxynucleotidyl transferase. Finally, using the Bi-MAb we developed a non-radioactive sandwich hybridization assay making use of oligonucleotide covalently bound to microwells and of synthetic oligonucleotide tailed with AAF-dGTP as a probe.
Auriol
J
J
Laboratoire de Prédéveloppement des Sondes, Institut Pasteur, Paris, France.
Chevrier
D
D
Guesdon
J L
JL
D000099:Acetoxyacetylaminofluorene / Q000378:metabolism; D000911:Antibodies, Monoclonal; D004247:DNA; D018736:DNA Adducts; D015342:DNA Probes; D004269:DNA, Bacterial; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D005456:Fluorescent Dyes; D007124:Immunoenzyme Techniques; D009169:Mycobacterium tuberculosis; D009693:Nucleic Acid Hybridization; D009713:Nucleotidyltransferases; D009841:Oligonucleotides; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D015342:DNA Probes; D004269:DNA, Bacterial; D003848:Deoxyguanine Nucleotides; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D000099:Acetoxyacetylaminofluorene; C029603:deoxyguanosine triphosphate; D004247:DNA; D009713:Nucleotidyltransferases