Collecting and Isolating Bovine DNA samples for testing

Since our project centers around developing a genetic testing system to detect genetic traits in dairy cows, it is inevitable that we need to develop procedures for both collecting samples from cattle and for extracting DNA from those samples. In the United States, any research that involves the use of vertebrate animals at a university must be done under the supervision of the Institutional Animal Care & Use Committee (IACUC). IACUC is responsible for ensuring that any work involving the use of animals maintains the highest standards in terms of animal welfare and maintenance when conducting that research. Before we began, we wrote a protocol for the collection of hair and buccal swabs from cattle and submitted those procedures to our institutions IACUC committee for approval.

To ensure the collection of sufficient DNA samples, we reached out to several cattle experts and researchers about different DNA extraction and collection methods. During our conversations with researchers Chad Dechow and Heather Husen we learned that both hair and saliva are excellent sources of DNA for genetic testing. Being unsure as to which type of sample would yield the best quality DNA, we elected to collect both to enable comparison.

We traveled to the Tauzel family Dairy farm, located approximately 20 minutes from our school to collect samples. Mr. Tauzel recommended that we collect samples from some calves, as opposed to adults, since calves are significantly smaller than full grown animals, reducing risk. Since our genetic testing system does not need samples from animals of a certain age, we agreed.

Figure 1: Visual representation of sample collection process to be used for genetic testing.

To obtain the DNA samples, we used a sterile buccal swab to gently remove cells from the inside cheeks of the calves’ mouths. The end of the swab was quickly detached and placed in a microcentrifuge tube for later use. The other samples collected were the hairs from the animal’s tail. For this method of collection, a few hairs were plucked from the tail and placed into a 50 mL conical tube for later use. All buccal and hair samples were labeled to indicate the animal they were obtained from and brought back to the taken from the calves were brought back and stored at -20° for later extraction of DNA.

Some of the processes of extracting DNA samples can be seen photographed below.

Figure 2: Team members Louise and Jazmine work to collect hair and buccal samples for DNA under the supervision of Team advisor Dr. Gallagher and local dairy farmer Mr. Tauzel.

DNA Extraction Using Cellulose Discs

Based on the methods from the paper, “Nucleic acid purification from plants, animals and microbes in under 30 seconds”

1. Preparation of 20mL (50µL/rxn) of Extraction Buffer:

   a. NOTE: Calculations with formula weights will be adjusted to match formula weights of the reagents on-bottle.

   • Weigh out Tris [pH 8.0] (50mM) (FW: 121.14g/mol)
     • (0.05M) x (0.02L) x (121.14g/mol)
       • 0.121g Tris (pH 8.0)
   • Weigh out NaCl (150mM) (FW: 58.44g/mol)
     • (0.15M) x (0.02L) x (58.44g/mol)
       • 0.175g NaCl
   • Weigh out PVP (2% PVP per 20mL solution)
     • 2% of 20mL: 0.4g PVP
   • Weigh out Tween-20 (1% Tween-20(viscous liquid) per 20mL solution)
     • 1% of 20mL: 0.2mL Tween-20

• Combine materials in a 50-100mL beaker. Add about 10-15mL of deionized water. Stir over a stir plate with stir bar.
• After solids reagents have fully entered the solution, transfer to a small graduated cylinder and add deionized water up to 20mL. Store in labeled 50mL (or similarly sized) bottle and store in fridge.

2. Preparation of 80mL (200µL/rxn) of Wash Buffer:

   a. NOTE: Calculations regarding formula weights will be adjusted once on-bottle formula weights of reagents are known.

   • Weigh out Tris [pH 8.0] (10mM) (FW: 121.14g/mol)
     • (0.01M) x (0.08L) x (121.14g/mol)
       • 0.097g Tris (pH 8.0)
   • Weigh out Tween-20 (1% Tween-20(viscous liquid) per 80mL solution)
     • 1% of 80mL: 0.8mL Tween-20

• Combine materials in a 50-100mL beaker. Add about 10-15mL of deionized water. Stir over a stir plate.
• After solids reagents have fully entered the solution, transfer to a small graduated cylinder and add deionized water up to 20mL. Store in labeled 50mL (or similarly sized) bottle and store in fridge.

3. To a 1.5mL microfuge tube, add a small amount of bovine hair or saliva. Add 50µL of Extraction Buffer to the tube.

4. Use a plastic pestle to crush the sample into the buffer.

5. Use a hole-puncher to punch No.1 Whatman’s Paper into a 3mm disc. Gently dip the disc into the 1.5 tube containing the tissue and buffer using forceps or similar tool. Leave disc in buffer for at least 3 seconds.

6. Carefully transfer disc from the tube containing the extraction buffer and tissue to a new tube containing 200µL of Wash buffer. Swirl gently for at least one minute.

7. Elute DNA step:

   a. Remove the disc from the buffer. Immerse in solution containing 45µL of Nuclease-free water and 5µL of dNTPs. Swirl gently for at least one minute

   i. The presence of dNTPs in solution further elutes DNA.

8. Check the concentration and purity of the extracted DNA using the NanoDrop.

   a. If the 260/280 measurement is greater than 1.7 (close to ~1.8), protein contamination is low enough to go through with an amplification (RPA) reaction on sample(s).

Resources:

Zou, Y., Mason, M. G., Wang, Y., Wee, E., Turni, C., Blackall, P. J., Trau, M., & Botella, J. R. (2017). Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLOS Biology, 15(11). https://doi.org/10.1371/journal.pbio.2003916