Native genomic blotting: a novel approach to mapping DNase I hypersensitive sites and protein-DNA interactions at high resolution.
We have developed a new high resolution method for screening 400-600 base pairs of DNA in chromatin for DNase I hypersensitive sites and protein-DNA interactions. By separating the DNA isolated from nuclease-digested nuclei in small, native polyacrylamide gels prior to electroblotting onto nylon membranes, we increased the resolution by greater than 3-fold as compared with the traditional approach whereby the nuclease-digested DNA is fractionated electrophoretically in agarose gels (11). In addition, our native genomic blotting method has the advantage of combining the ability of the traditional agarose approach to detect DNase I hypersensitive sites, with the genomic sequencing method (2), where individual protein-DNA contacts can be observed. Native genomic blotting therefore permits for the first time the display of DNase I hypersensitive sites and protein-DNA interactions at high resolution on the same autoradiograph. This method allows us to investigate a new level of chromatin structure and to therefore obtain better insight into levels of gene structure, organization and gene regulation.
Pauli
U
U
University of Florida, College of Medicine.
Chrysogelos
S
S
Stein
J
J
Stein
G
G
D001345:Autoradiography; D004247:DNA / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000235:genetics; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D009693:Nucleic Acid Hybridization; D009710:Nucleotide Mapping / Q000379:methods*
D015342:DNA Probes; D004268:DNA-Binding Proteins; D006657:Histones; D004247:DNA; D003850:Deoxyribonuclease I