Fluorescence-Based Sorting of Individual Virus Particles for Genomic Analysis

Take a suspension containing a diverse mixture of viruses, along with contaminants such as defective viral particles lacking nucleic acids.

These viruses are pre-stained with a nucleic acid–binding fluorescent dye to distinguish viruses with genomes from contaminants.

Pass the suspension through a fluorescence-activated cell sorter, where each particle is analyzed for light scatter and fluorescence intensity.

The light scatter signal reflects particle size, while the fluorescence intensity indicates nucleic acid content.

Exclude contaminants that show no fluorescence and aggregates that display abnormally high fluorescence.

Select single virus particles that contain a genome and sort them directly onto an agarose-coated chambered slide for immobilization.

Overlay each well with additional agarose to embed the viruses.

These are now ready for single-virus genome amplification and analysis.

To sort viruses using a flow cytometer equipped with custom forward scatter photomultiplier tubes or FSC-PMT. Begin by diluting the viral particles in 0.1 micron filtered Tris-EDTA. For a mixed assemblage containing T4 and lambda phage particles, set the FSC-PMT to 1000 and the SSC to 200 to maximize the