Dot Blot Assay to Quantify Viral Genome

NOTE: Recipes for the solutions and buffers needed for this protocol are provided in Table 1.

1. Dot blot

1. Preparation of plasmid DNA standards
   1. Dilute AAV vector genome plasmid DNA to 10 ng/µL in water or Tris-HCl buffer (10 mM, pH 8.0). Take 25 µL of this dilution and digest with an appropriate restriction enzyme for 1 h to linearize the plasmid DNA in a reaction volume of 50 µL.
      NOTE: We make a duplicated set of digestion (see step 1.4.1). The appropriate enzyme should be one that cuts the plasmid DNA outside the dot blot probe-binding region. For convenience, the diluted plasmid DNA can be aliquoted (25 µL/tube) and stored frozen at -20 °C for future use. Digest the plasmid DNA while the tubes are shaking in step 5.1. Do not over-digest the plasmid DNA standard.
   2. Add 450 µL of water or TE to the tube containing the digested plasmid DNA standard and mix thoroughly. Transfer 70 µL of this mixture to a new 1.5 mL microcentrifuge tube with 1,330 µL of water or TE to make a diluted plasmid standard (25 pg/µL).
   3. Follow Table 1 to create a set of two-fold serial dilutions (600 µL/tube). Mix the dilutions thoroughly by vortexing for 5 s.
2. Denaturing of standards and viral DNA samples
   1. Add 600 µL of 2x Alkaline Solution to each standard dilution. Mix well by vortexing for 5 to 10 s. Incubate at room temperature for 10 min.
   2. Add 120 µL of 2x Alkaline Solution to each viral DNA sample. Mix well by vortexing for 5 to 10 s. Incubate at room temperature for 10 min.
3. Setting up the dot blot apparatus
   1. Using scissors, cut a blotting (e.g., zeta-probe) membrane to an appropriate size for the number of samples and standards. Soak the membrane with water for 10 min before placing it on a dot blot apparatus. Cover unused wells on the membrane apparatus.
      NOTE: Handle the membrane with clean tweezers. To cover empty wells, the light blue protection sheet that comes with the membrane can be used. Do not allow the membrane to dry prior to binding samples and standards. For more details on the assembly and use of the apparatus, please refer to the user manual.
   2. Add water to the wells to which samples will be loaded. Apply vacuum and pull water through the wells to check for errors and retest when fixed. Re-tighten the screws while applying vacuum to ensure tight sealing.
      NOTE: Incomplete sealing may cause sample leakage between the wells.
   3. Once water is completely pulled through, release the vacuum completely (i.e., the vacuum manifold should be open to air pressure).
4. Loading standards and samples to the dot blot apparatus
   1. Apply 400 µL 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. Apply 200 µL/well of each viral DNA sample.
      NOTE: Using the remaining ~40 µL of denatured samples, diluted samples can be prepared (e.g., 10-fold diluted samples using 20 µL of the sample plus 180 µL of 1x Alkaline Solution) and blotted if necessary.
   2. Apply a gentle vacuum to pull the DNA solutions through the well.
      NOTE: Vacuum pressure needs to be adjusted by partially opening a three-way valve so that the vacuum pressure is applied to the dot blot apparatus as well as the atmosphere (i.e., with the stopcock arms positioned at an approximately 45° angle where it makes a louder suction noise).
   3. Once all the wells have emptied, release the vacuum by adjusting the three-way valve. Add 400 µL of 1x Alkaline Solution to each well that contained standards and samples. Wait for 5 min before re-applying the vacuum to empty the wells.
   4. Re-apply the vacuum in the same way.
   5. Disassemble the dot blot apparatus under the vacuum, 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 2x SSC.
   6. UV-crosslink the blotted DNA to the membrane with an appropriate UV crosslinker; the membrane is now ready for hybridization.
      NOTE: Wet membranes can be used for UV crosslinking. The dried, UV-crosslinked membranes can be stored at room temperature. Further information can be found in the Table of Materials.

2. Hybridization and washing

1. Warm up the Hybridization Buffer in a 65 °C water bath.
2. Enzymatically label a DNA probe with radioactive α-³²P dCTP and purify it using commercially available kits according to the manufacturer's recommendation.
   NOTE: We use a probe of 0.5–2.0 kb in length from an enhancer-promoter region or a protein-coding sequence in the viral genome. Although this protocol utilizes ³²P-labeled radioactive probes for signal detection, non-radioactive chemiluminescent or fluorescent probes can also be used (please refer to the Discussion section).
3. Place the membrane in a hybridization bottle with the DNA-bound side up, rinse the membrane with 5 mL prewarmed Hybridization Buffer, and discard the buffer. Then add 10 mL of prewarmed Hybridization Buffer and place the bottle in a rotating hybridization oven set at 65 °C. Rotate for ≥5 min.
4. Heat-denature 20 µL of 10 mg/mL sheared herring or salmon sperm DNA solution and the ³²P-labeled probe (≥10⁷ cpm) for 5 min by placing the tubes on a heat block set at 100 °C. Then snap-chill them on ice for ≥2 min, spin briefly, and keep them on ice until used.
   Caution: For radioactive DNA probes, 1.5 mL tubes with screw caps and O-rings must be used.
5. Quickly add the denatured sperm DNA and radioactive probe to the Hybridization Buffer in the hybridization bottle and shake the bottle for 10 s to mix. Return the bottle to the 65 °C oven and incubate the bottle with rotation at 65 °C for ≥4 h.
6. Once hybridization is complete, stop the rotation, remove the hybridization bottle, and then pour the radioactive probe solution into a 50 mL conical tube with a leak-proof plug seal cap. Store the probe in an appropriate container placed in a refrigerator designated for radioactive materials.
   NOTE: Used Hybridization Buffer with a probe that is stored at 4 °C can be re-used at least 5 times by placing the 50 mL conical tube with a leak-proof plug seal cap that contains Hybridization Buffer in a 100 °C water bath for 5 min.
7. Wash the membrane with Wash Buffer heated to 65 °C. Add 20 to 30 mL of Wash Buffer to the hybridization bottle and rotate for 5 min. Repeat this wash 2 more times.
   NOTE: Measure the radioactivity of wash solutions and record it if required by the local institute.
8. While washing the membrane, place a phosphor imaging screen on an image eraser for 5 min.
9. After the third wash, remove the membrane from the hybridization bottle, remove the excess buffer on the membrane with paper towels, and place the membrane in a clear plastic paper holder. Check radioactive signals on the membrane using a Geiger counter. Expose the erased phosphor imaging screen to the membrane for 10 min to overnight, depending on the signal intensity.
10. Scan the screen using a phosphor image scanning system and obtain the data on the signal intensity of each dot.

Table 1: Quantitative dot blot plasmid DNA standards. Necessary volumes of 25 pg/µL plasmid DNA standard solution and water or TE to prepare plasmid DNA standards by two-fold serial dilutions (600 µL/tube) are shown. The numbers in the plasmid DNA standard column (0.039 to 5 ng) are the quantity of DNA in 200 µL of each plasmid DNA standard solution.