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.
Development of Field-friendly DNA extraction methods
Our team collected tissue and saliva samples from local farm cows to have its DNA extracted. Most standard DNA extraction protocols count for centrifugation that is not easily available on the field. For Flappase to detect the A1/A2 SNP, DNA must be amplified. For DNA to be amplified, it must be extracted (Figure 3).
Figure 3: Process of converting extracted DNA samples from Bos taurus to amplified DNA with an SNP detectable by Flappase Assay.
Using the protocol we developed, an extraction was performed on two sets of calf hair samples. 10-15 follicles, approximately 1.5-3cm in length, of hair tissue per sample from calves, “Elmer” and “Booki”, were used to extract DNA. The concentration and purity of the extracted DNA was measured using the NanoDrop One. The following are concentration and absorbance readings for both samples:
| Cow | DNA Concentration | A260/230 | A260/280 |
|---|---|---|---|
| Elmer | 965.4 ng/µL | 1.7 | 2.05 |
| Booki | 46.5 ng/µL | 0.71 | 1.05 |
The concentrations of both eluted samples were high enough (>20ng/µL) for their absorbance ratios to be used (1). The A260/280 ratio of eluted “Elmer” was within the range of good quality for DNA (1.7-2.0). The A260/230 ratio was also above recommended minimums for quality DNA (>1.5) (2). With a concentration at nearly 1 µg/µL, we plan to prepare 50 ng/10µL solutions of eluted “Elmer” and run samples on an agarose gel, using ethidium bromide to visualize the purity of the DNA. Many contaminants, such as RNA, will appear at a lower molecular weight on the gel (2).
While the concentration of eluted “Booki” was still above the usable level, both A260 absorbance ratios in the sample were too low to be indicative of quality DNA, as more light would have been absorbed at this 260 nm wavelength if more DNA were present in the sample (2). The most probable cause of this low absorbance was likely the addition of too much extraction buffer at the beginning of the procedure for this sample. The increased buffer volume caused extracted DNA to be dispersed more openly in the solution and less concentrated.
Some modifications to the protocol are being considered after this extraction trial:
With the difficulty of gathering hair samples uniformly into a microfuge tube, it may be advised to saturate the hair with a small amount of buffer or water into the vessel it came from.
- This will cause the hair to stick to itself, allowing for ease of transfer.
dNTPs were added to the water used for elution at a concentration of 1mM. Different concentrations of dNTPs may be tried to optimize elution.
- It has been shown in other systems that dNTPs can catalyze the elution of the DNA from the cellulose. However, if there is too much cellulose in the reaction, it can sequester all the dNTPs in solution and interfere with elution and downstream amplification reactions (1). Increasing the concentration of dNTPs during elution could increase DNA elution.
Only DNA from hair tissue was eluted in these trials. To visualize extraction results from multiple tissue sources, DNA from saliva samples that were collected by the team will be extracted in the next trial.
Amplification of target gDNA sequences for detection
In order to detect a polymorphism within a gene using our genetic testing system, it will be necessary to amplify the signal. The simplest way to accomplish this is to amply the signal by starting with more copies of the DNA sequence containing the polymorphism to be detected. Last year, the team decided to use Recombinase Polymerase Amplification (RPA) to do this.
RPA has advantages over standard PCR, which make use of this technique to amplify our target gene for its more field-friendly implementation. First, most farms lack the thermal cycler that is required for PCR amplification of DNA. With RPA, the thermal cycling required to separate and anneal DNA is replaced by enzymatic activity. Second, RPA reactions are held at a constant temperature between 37ºC and 42ºC, which is easier to provide. Finally, the RPA reaction time is significantly lower than that of standard PCR. RPA shows to be a more suitable option for implementation in a farm, as it requires fewer equipment and takes less time than standard PCR.
Figure 4: Comparison of the annealing steps of RPA and PCR. The thermal denaturation and annealing steps of PCR are replaced by the presence of enzymes in RPA that help the primers anneal to their complimentary target DNA (4).
Last year, the team created several sets of primers to be used to selectively amplify a fragment of the beta-casein gene. This fragment contains the SNP that creates the A1 and A2 alleles. The team also began working on optimizing the RPA protocol.
Optimization of Selected Primers for DNA amplification
Last year, three pairs of forward and reverse primers were selected as candidates for the amplification of the Exon 7 region of the CSN2 gene in Bos taurus. Forward primers 2F, 4F, and 18F, and reverse primers 6R, 10R, and 11R were tested at under varying pairings using Recombinase Polymerase Amplification (RPA). Pairs 2F,10R and 4F,11R were selected, showing bands in the desired molecular weight. 4F,11R and 2F,10R primer pairs were subjected to temperature optimization RPA reactions at 37ºC and 42ºC. Reactions showed that the 37ºC runs best suited the 2F,10R pair, and the 4F,11R worked effectively at both 37ºC and 42ºC.
This year, temperature optimization of the two pairs was repeated with a fresh RPA kit to confirm the temperature-specific activity of primer pairs.
Figure 5: Temperature Optimization trials for primer pairs 2F,10R and 4F,11R. RPA reaction were conducted at the indicated temperatures for 40 minutes. Reaction products were subjected to 0.8% agarose gel electrophoresis. Size of expected products to be at ~300bp.
37ºC and 42ºC reactions showed bands in the expected product size, (~300bp) for both 2F,10R and 4F,11R. Observing a notable difference of band intensity between primers and temperatures, we decided to test primer activity against varying volumes of primer per reaction. (Figures 2 and 3)
Figure 6: RPA primer volume trials using differing volumes of 2F,10R primer pair. Samples were incubated at 37°C for 30 minutes. Reaction products were subjected to 0.8% agarose gel electrophoresis. Size of expected products to be at ~300bp.
Figure 7: RPA primer volume trials using differing volumes of 4F,11R primer pair. Samples were incubated at 42°C for 20 minutes. Reaction products were subjected to 0.8% agarose gel electrophoresis. Size of expected products to be at ~300bp.
For both primers, reactions using only 2.4µl of primer pair yielded better results than reactions with the larger 4.8µl volume. With this in mind, we intend to optimize the reaction time for both pairs and further characterize a reaction scheme with optimal time, temperatures, and primer volumes with respect to each pair.
References
- Zou, Y., Mason, M. G., Wang, Y., Wee, E., Turni, C., Blackall, P. J., Trau, M., & Botella, J. R. (2017, November). Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLOS Biology. Retrieved October 18, 2021, from https://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.2003916#pbio.2003916.ref065.
- Koetsier, G. (2019). A Practical Guide to Analyzing Nucleic Acid Concentration and Purity with Microvolume Spectrophotometers. Technical Note. Retrieved October 18, 2021, from https://www.neb.com/-/media/nebus/files/application-notes/technote_mvs_analysis_of_nucleic_acid_concentration_and_purity.pdf?rev=c24cea043416420d84fb6bf7b554dbbb&hash=475700CBD975A281D7C362992137F59A.
- Oswald, N. (2020, April 8). Quick reference: Determining DNA concentration & purity. Bitesize Bio. Retrieved October 18, 2021, from https://bitesizebio.com/25270/quick-reference-determining-dna-concentration-purity/.
- Twist-DX. (n.d.). Recombinase Polymerase Amplification, or RPA, is the breakthrough, isothermal replacement to PCR. StackPath. https://www.twistdx.co.uk/en/rpa.