title: A dual color Southern blot to visualize two genomes or genic regions simultaneously.
abstract: This report describes the development of a novel dual color Southern protocol to visualize two distinct genomes or genic regions simultaneously on a single Southern blot. The blot is developed with IRDye-conjugated antibody (Ab) and streptavidin that recognize digoxigenin (Dig)- or biotin-labeled probes, respectively and visualized on an infrared imager. This protocol was validated by visualizing viral and host genomes of human cytomegalovirus (HCMV)-infected human fibroblasts. This technique utilizes extremely sensitive fluorescent imaging, allowing the detection of nanogram quantities of DNA, as opposed to microgram quantities needed in Southerns using radioactively labeled probes, and eliminates the inherent loss in signal after stripping and reprobing a Southern blot. The probes are labeled with non-radioactive Dig and biotin and can be stored for extended periods of time. This protocol will aid in studies of any system with two genomes, such as cells infected with numerous types of microorganisms (virus/parasites/bacteria), or studies of mitochondrial and nuclear DNA within the same cells.
authors.affiliation: Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID, USA.
authors.forename: Anamaria G
authors.initials: AG
authors.lastname: Zavala
authors.affiliation: Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID, USA.
authors.forename: Amit S
authors.initials: AS
authors.lastname: Kulkarni
authors.affiliation: Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID, USA. Electronic address: lfort@uidaho.edu.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Fortunato
mesh_terms: D015139:Blotting, Southern / Q000379:methods*; D003587:Cytomegalovirus / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D016678:Genome / Q000235:genetics*; D006801:Humans
chemical_list: D005456:Fluorescent Dyes; D004247:DNA
keywords: DNA analysis; Dual-color Southern; IRDyes; Li-Cor Odyssey; Non-radioactive Southern; Simultaneous two-genome detection in infected cells