A dual color Southern blot to visualize two genomes or genic regions simultaneously

Anamaria G Zavala; Amit S Kulkarni; Elizabeth A Fortunato

doi:10.1016/j.jviromet.2013.12.019

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A dual color Southern blot to visualize two genomes or genic regions simultaneously

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A dual color Southern blot to visualize two genomes or genic regions simultaneously

Anamaria G Zavala, Amit S Kulkarni and Elizabeth A Fortunato

Journal of virological methods, Vol.198, pp.64-68

03/2014

DOI: https://doi.org/10.1016/j.jviromet.2013.12.019

Handle:

https://hdl.handle.net/2376/108388

PMCID: PMC4010645

PMID: 24389128

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Abstract

DNA - genetics

Blotting, Southern - methods

Cytomegalovirus - chemistry

Cytomegalovirus - genetics

Fluorescent Dyes - chemistry

Humans

Genome - genetics

Fibroblasts - chemistry

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.

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Title: A dual color Southern blot to visualize two genomes or genic regions simultaneously
Creators: Anamaria G Zavala - Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID, USA
Amit S Kulkarni - Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID, USA
Elizabeth A Fortunato - Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID, USA. Electronic address: lfort@uidaho.edu
Publication Details: Journal of virological methods, Vol.198, pp.64-68
Academic Unit: School of Biological Sciences
Publisher: Elsevier; Netherlands
Number of pages: 5
Grant note: P20 GM103408 / NIGMS NIH HHS P20 RR016454 / NCRR NIH HHS R21 AI051463 / NIAID NIH HHS R01 AI51463 / NIAID NIH HHS R01 AI051463 / NIAID NIH HHS P20 RR015587 / NCRR NIH HHS
Identifiers: 99900547042601842
Language: English
Resource Type: Accepted manuscript