Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-06-20
1998-05-05
Degen, Nancy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 29, 436124, 436164, 436172, C12Q 168, C12Q 102, G01N 2175, G01N 2176
Patent
active
057472580
ABSTRACT:
A method for detecting halogenated precursors incorporated into DNA is presented. The method is based on the selective photolysis of DNA by ultraviolet (UV) light at the sites of an incorporated halogenated precursor, such as the thymidine base analogs 5-bromo-2-deoxyuridine (BrdUrd), 5-iodo-2-deoxyuridine (IdUrd), 5-fluoro-2-deoxyuridine (FdUrd), or 5-chloro-2-deoxyuridine (CldUrd). The 3'-hydroxyl termini of the DNA single strand breaks generated during photolysis may be marked directly or indirectly with a fluorescent label. The DNA termini are directly labeled with fluorochrome-conjugated deoxyuridine triphosphate (dUTP) catalyzed by exogenous terminal deoxynucleotidyl transferase or DNA polymerase (nick translation system). The DNA termini are indirectly labeled with either biotin- or digoxygenin-conjugated dUTP; the incorporated biotin or digoxygenin is then detected following binding of fluorochrome-conjugated avidin or anti-digoxygenin antibody, respectively. The labeled DNA may be analyzed in situ by flow cytometry or fluorescence microscopy. Alternatively, the DNA may be isolated and analyzed by conventional methods, including gel electrophoresis and blotting assays, prior to marking with a flourescent label. The method does not require denaturation of the DNA and may be used with cells in suspension, thin tissue sections, bacteria, and viruses. The method has application in the analysis of cell proliferation and genotoxicity tests.
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Darzynkiewicz Zbigniew D.
Li Xun
Melamed Myron R.
Traganos Frank N.
Degen Nancy
New York Medical College
Sommer Evelyn M.
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