Dot Blot Assay to Quantify Viral Genome

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.

To carry out the dot blot assay, prepare plasmid DNA standards by diluting AAV vector genome plasmid DNA to 10 nanograms per microliter in water or Tris-HCl buffer. Transfer a 25-microliter aliquot of the diluted plasmid DNA to a fresh tube, and linearize it with an appropriate restriction enzyme in a 50-microliter reaction volume for one hour.

Add 450 microliters of water or TE to the tube containing the digested plasmid DNA standard, and mix thoroughly. Then, transfer 70 microliters of this mixture to a new 1.5-milliliter microcentrifuge tube with 1,330 microliters of water or TE to make a diluted plasmid standard.

To set up the dot blot apparatus, using scissors, cut the blotting membrane to an appropriate size for the number of samples and standards. Soak the membrane with water for 10 minutes, before placing it on the dot blot apparatus. Then, cover the unused wells. Add water to the wells to which the samples will be loaded. Apply a vacuum, pull the water through the wells, check for errors, and then re-tighten the screws. Re-test if necessary.

Apply 400 microliters of each diluted plasmid DNA standard to each well and run four lanes of standard dilutions. Use two separate aliquots of the standard digest, and load each in duplicate. Then, apply 200 microliters per well of each viral DNA sample. Apply a gentle vacuum to pool the DNA solutions through the wells. Then, once all the wells have emptied, release the vacuum by adjusting the three-way valve.

Add 400 microliters of 1X alkaline solution to each well. Then, wait for five minutes before reapplying vacuum to empty the wells. Re-apply the vacuum and disassemble the dot blot apparatus. Then, remove the membrane and rinse it with 2X SSC.

Place the membrane on a clean paper towel, with the DNA-bound side facing up, to remove excess buffer. Use an appropriate UV crosslinker to UV-crosslink the blotted DNA to the membrane. The membrane is now ready for hybridization.

Place the membrane in a hybridization bottle with the DNA-bound side up. Rinse the membrane with 5 to 10 milliliters of pre-warmed hybridization buffer, and discard the buffer. Then, add 10 milliliters of pre-warmed hybridization buffer, and place the bottle in a rotating hybridization oven set to 65 degrees Celsius. Rotate the bottle for at least five minutes.

Quickly add the denatured sperm DNA and radioactive probe to the hybridization buffer in the bottle, and shake it for 10 seconds to mix. Return the bottle to the 65-degree Celsius oven and incubate it with rotation at 65 degrees Celsius for at least four hours.

Once hybridization is complete, stop the rotation, remove the hybridization bottle, and then pour the radioactive probe solution into a 50-milliliter conical tube with a leak-proof plug seal cap. To wash the membrane, add 20 to 30 microliters of pre-warmed wash buffer to the hybridization bottle, and rotate it for five minutes. Repeat this wash two more times.

After the third wash, remove the membrane from the hybridization bottle, and with paper towels, remove the excess buffer from the membrane. Then, place the membrane in a clear plastic paper holder. With a Geiger counter, check the radioactive signals on the membrane.

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After exposing the erased phosphor imaging screen to the membrane, scan the screen using a phosphor image scanning system, and obtain the data on signal intensity of each dot.