Native genomic blotting: high-resolution mapping of DNase I-hypersensitive sites and protein-DNA interactions

Journal Article · Fri Jan 01 04:00:00 EST 1988 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.85.1.16· OSTI ID:6464732

Pauli, U; Chrysogelos, S; Stein, J; Stein, G

DNase I-hypersensitive sites are observed in the promoter regions of actively expressed genes, potentially active genes, and genes that were once active. The authors have developed an approach that greatly increases the resolution for mapping these sites by electrophoresing genomic DNA on native polyacrylamide gels prior to electroblotting and hybridization. This improved method has been used to scan the promoter and coding region of a cell-cycle-dependent human histone H4 gene with an accuracy of +/- 5-10 base pairs. Protein-DNA interactions can be seen in the autoradiograph as light areas and DNase I-hypersensitive sites as dark bands. Therefore, this method provides a rapid and relatively simple means to accurately localize protein-DNA interactions as well as DNase I-hypersensitive sites, thus directly displaying DNase I hypersensitivity and protein-DNA complexes on one autoradiograph. It also potentially allows the analysis of small changes in DNase I-hypersensitive sites under various biological conditions. With this technique rather large regions of DNA can be screened to determine areas that should be analyzed by more sophisticated methods, such as genomic sequencing or gel retardation assays.

• Research Organization: Univ. of Florida College of Medicine, Gainesville (USA)
• OSTI ID: 6464732
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 85:1; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AUTORADIOGRAPHY
CHEMICAL REACTIONS
CHROMATIN
CROSS-LINKING
DNA-ASE
ELECTROMAGNETIC RADIATION
ELECTROPHORESIS
ENZYMES
ESTERASES
GENE REPRESSORS
GENES
GENETIC MAPPING
HELA CELLS
HISTONES
HYBRIDIZATION
HYDROLASES
MAPPING
MOLECULAR STRUCTURE
NUCLEOPROTEINS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
POLYMERIZATION
PROTEINS
RADIATIONS
ULTRAVIOLET RADIATION