Encyclopedia of Experiments: Biological Techniques
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Dot Blot Assay to Quantify Viral Genome - ExperimentTranscript
To quantify viral DNA using the dot blot assay, begin with an isolated viral DNA suspension. Treat with an alkaline solution to denature the double-stranded DNA into single-strands for subsequent hybridization.
Assemble the dot blot apparatus with a pre-wetted nylon membrane. Load denatured viral DNA and linearized standard plasmid DNA solutions into the wells. Apply a suitable vacuum, facilitating effective negatively-charged viral single-stranded DNA transfer and binding onto the positively-charged membrane surface through electrostatic interactions, forming dot patterns.
Following the run, wash the membrane with sodium citrate buffer, helping improve the assay sensitivity. Post-drying, expose the membrane to ultraviolet light, crosslinking the blotted DNA to the membrane.
Add a heat-denatured, non-heterologous DNA fragment and viral genome-specific radioactive phosphorus-labeled DNA probe suspension in hybridization buffer to the membrane. Incubate, facilitating hybridization.
The DNA fragments act as blocking agents, binding to the remaining membrane regions, minimizing non-specific probe binding. The radioactive probe hybridizes with the complementary sequence on the viral single-stranded DNA.
Wash with wash buffer to remove unhybridized probes. Using the phosphor image scanning system, quantify the radioactive signals emitted by the radioactive probes hybridized to the viral genome on the membrane, to obtain dot blot signals.
From the standard curve, the signal intensity of each dot on the membrane can be used to calculate the amount of viral DNA in the sample.
