JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:35 min • September 30th, 2026
Take culture supernatants containing progeny virions released from host cells infected with either a test HIV-1 variant or a wild-type control.
The test virus carries a gene variant that alters replication efficiency and affects progeny virion production.
Add the supernatants to a reverse transcription mixture containing a radiolabeled nucleotide, an unlabeled nucleotide, an RNA template, a primer, cofactors, detergent, and stabilizers.
Incubate to allow template–primer complex formation while the detergent lyses virions, releasing reverse transcriptase, or RT.
RT synthesizes DNA from the template–primer complex, incorporating the radiolabeled nucleotide.
Transfer samples to a nucleic acid–binding membrane and air-dry to immobilize the radiolabeled DNA.
Wash repeatedly with buffer and ethanol under agitation to remove unbound components.
Air-dry and wrap the membrane. Expose it to a radiation-sensitive screen and detect signals using a phosphorimager.
A reduced signal in the test indicates lower RT levels, reflecting decreased virion production compared to the control.
In this procedure, add one to two microliters of 10 millicuries per milliliter of dTTP and four microliters of one molar di
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