Immunoseparation and immunodetection of nucleic acids labeled with halogenated nucleotides.
A novel methodology for labeling, isolation, and detection of nucleic acids is described. Nucleic acid isolation is based on in vivo or in vitro incorporation of BrU or BrdU to either RNA or DNA, respectively, followed by immunoprecipitation of the labeled nucleic acid utilizing anti-BrdU MoAb, which crossreacts with BrU, attached to solid particles. Filter-bound bromine-labeled DNA or RNA was detected by immunoblotting with anti-BrdU MoAb, by a combined Southern/Western or Northern/Western approach, respectively. This method was applied to isolate and detect rRNA and mRNA from human cells, plasmid DNA from bacterial cells, and in vitro synthesized DNA. Newly transcribed BrU-labeled mRNA was recovered from the immunoprecipitates and analyzed by RT-PCR to study phorbol ester-mediated regulation of interleukin 1 gene transcription in human leukemic HL-60 or lymphoma U937 cells. The plasmid DNAs were isolated by immunoprecipitation from transformed bacterial cultures that were grown in the presence of BrdU and were detected immunochemically on filters. Likewise, the products of RT-PCR and Klenow polymerase-catalyzed DNA synthesis in which dTTP was replaced with BrdUTP were detected by immunoblotting. Since the method allows one to selectively separate or detect nucleic acids only synthesized during a pulse of the precursor, it can uniquely be used to identify nascent gene transcripts or the transcripts synthesized within specific time windows, e.g., after induction of differentiation, carcinogenesis, or drug treatment, and distinguish such transcripts from preexisting ones. In addition, this approach offers a simple and inexpensive alternative for preparing labeled DNA as well as RNA probes for use in a variety of hybridization protocols. Due to the low toxicity of BrU and BrdU, this approach can be used in analysis of gene transcription or DNA replication in vivo.
Haider
S R
SR
The Cancer Research Institute, New York Medical College, Valhalla 10595, USA. raza_haider@nymc.edu
Juan
G
G
Traganos
F
F
Darzynkiewicz
Z
Z
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001973:Bromodeoxyuridine; D001976:Bromouracil / Q000031:analogs & derivatives; D003429:Cross Reactions; D004269:DNA, Bacterial / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D003856:Deoxyuracil Nucleotides; D018922:HL-60 Cells; D006801:Humans; D015336:Molecular Probe Techniques; D009696:Nucleic Acids / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011233:Precipitin Tests; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000031:analogs & derivatives*
D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D003856:Deoxyuracil Nucleotides; D009696:Nucleic Acids; C032549:bromodeoxyuridine triphosphate; D001976:Bromouracil; D012313:RNA; D001973:Bromodeoxyuridine; C006824:5-bromouridine; D014529:Uridine