TY - JOUR
T1 - A dual color Southern blot to visualize two genomes or genic regions simultaneously
AU - Zavala, Anamaria G.
AU - Kulkarni, Amit S.
AU - Fortunato, Elizabeth A.
PY - 2014/3/15
Y1 - 2014/3/15
N2 - 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.
AB - 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.
KW - DNA analysis
KW - Dual-color Southern
KW - IRDyes
KW - Li-Cor Odyssey
KW - Non-radioactive Southern
KW - Simultaneous two-genome detection in infected cells
UR - http://www.scopus.com/inward/record.url?scp=84892632380&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2013.12.019
DO - 10.1016/j.jviromet.2013.12.019
M3 - Review article
C2 - 24389128
AN - SCOPUS:84892632380
SN - 0166-0934
VL - 198
SP - 64
EP - 68
JO - Journal of Virological Methods
JF - Journal of Virological Methods
ER -