Visualization of DNA replicons by image cytometry
- AMC ImmunoDiagnostics, The Woodlands, TX (United States)
Replication of DNA in eukaryotic organisms proceeds bidirectionally along the double helix in replicon substructures. The process can be visualized by autoradiography of spreads of genomic DNA from lysed, whole cells which have been incubated with radioactive DNA precursors. The objective of the present study was to develop techniques to measure DNA strand initiation and elongation using immunofluorescence and image cytometry. Peripheral blood lymphocytes were cultured for 3 days with PHA and then pulsed for 5 or 15 minutes with iododeoxyuridine. Chromatin spreads were then produced on microscope slides by lysing with detergent and slides were immunofluorescently stained by an indirect anti-BrdU technique. Individual replicons of mammalian cells were visualized by immunofluorescence at high resolution and digital images were analyzed to determine the rates of elongation as well as initiation parameters. Elongation rates by the method were approximately 1 [mu]M/min. The methods are applicable to visualization and quantitation of the effects of radiation or other agents on DNA damage or repair.
- OSTI ID:
- 6824046
- Report Number(s):
- CONF-9303114-; CODEN: CYTODQ; TRN: 93-010281
- Journal Information:
- Cytometry (Baltimore); (United States), Vol. 6; Conference: 16. congress of the International Society for Analytical Cytology, Colorado Springs, CO (United States), 21-26 Mar 1993; ISSN 0196-4763
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
REPLICONS
BIOLOGICAL RADIATION EFFECTS
IMMUNOASSAY
DNA
DNA REPAIR
FLUORESCENCE
GENES
TOXICITY
BIOASSAY
BIOLOGICAL EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
LUMINESCENCE
NUCLEIC ACIDS
ORGANIC COMPOUNDS
RADIATION EFFECTS
REPAIR
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture
550200 - Biochemistry