Optimization of Selected Primers for Bos taurus 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 1: C for primers 2F 10R and 42°C for primers 4F 11R for 40 minutes. Reaction products were subjected to 0.8% agarose gel electrophoresis. Size of expected products to be at ~300bp.
Both 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 2: RPA 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 ~100bp.
Figure 3: RPA 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.
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 4).
Figure 4: Process of converting extracted DNA samples from Bos taurus to amplified DNA with an SNP detectable by Flappase Assay.
After researching several DNA extraction methods with no centrifugation step, we found a method involving the use of cellulose. Subsequent extractions will be tested. Cow samples will be ground up with a pestle in the presence of a buffer, cellulose-based paper will be introduced to the sample and washed using a different buffer. The extracted DNAs purity will be tested using a NanoDrop before performing RPA reactions on samples.
Additional References
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