MATERIALS & METHODS
This canine experiment took several lab periods and different protocols. Below is an overview of the protocols, with in-depth details to follow.
Collection of Dog DNA Samples from Classmates Dogs
Perform PCR for DNA Sequencing for SNPs
Perform PCR for DNA Sequencing for MCR1 and Our Chosen SNPs
DNA Analyze - Observing the Gels and Data Collection
1. DNA Collection
We first started with needed 30 cheek swab samples of 15 dogs of different breeds and morphological traits from dogs of students in our laboratory class to observe the differences.
2. First Lab of Canine Experiment - DNA Isolation
In the first lab period involving the canine experiments, we obtained our samples of the dog DNA. Our lab group had samples M1, M2, L1, L2. We followed the protocol for isolating the DNA with QIAamp DNA mini kits from buccal swabs. We lysed the cells, the precipitated the DNA so that we could collect the DNA molecules on the spin column. Next, we washed the NDA on the spin column. After, we eluted the sample and then collected the final purified and isolated DNA. Lastly, we checked the quantity and quality of our DNA samples using a NanoDrop by observing the concentration and absorbance ratios. Our results and data collected from our samples are shown in Table 1.
3. Second Lab of Canine Experiment - Designing dCAPS PCR Primers
In this lab period, we chose the specific morphological traits of dogs that we wanted to analyze, for later use on our dog samples collected and isolated in the previous lab periods. To do this, we had the design dCAPS PCR primers since this would allow us to view which samples have which polymorphism associated with the traits -- such as D1 primer and its SNP for [C/T]. To do so, we followed the extensive protocol given by the “dCAPS PCR Primer Design Protocol” file that was provided on Moodle, and used the website “dCAPS Finder 2.0” [http://helix.wustl.edu/dcaps/dcaps.html] . Yet, before we could do this, we had to enter our SNP data according to its specific format -- which required less than 60 characters and no non-[ATCG] characters. We created a table of all our information of the seven SNPS that the professor wanted us to use (Table 2), and well as another table of the information regarding our particular SNP that we chose -- herding (Table 3).
4. Third Lab of Canine Experiment - DNA Sequencing / PCR of SNP Markers
In this experiment, we ran PCR experiments to help us genotype each dog according to the SNPs that we worked on the week before which include DS 1 through 7 and also two control PCR reactions as well. The controls (a positive and a negative) were to make sure that the restriction digest worked and to create a band on our gels that we could compare each sample run to. We chose our dog sample that had the highest quality of DNA from our results from the DNA isolation - which was L2 and M1 - and followed the “Biology 284 - PCR of SNP Markers” protocol for this experiment. The SNP that we were given was DS7 for low weight. Yet, when creating the 14 tubes from the DNA samples, we ran our of L2 and had to use the lower quality L1 for the rest of the tubes. At the end of the period, we turned in 7 sample tubes of L2/L1 and M1 to Dr. Loomis for DNA sequencing of the MC1R gene, which will identify the color coat of the dogs. (Her procedure is stated in the next section.)
5. Fourth Lab of Canine Experiment - DNA Sequencing / PCR of MC1R and PCR of Our Chosen SNP Markers
Dr. Loomis performed PCR on the dog DNA samples were extracted and saved for her from last week. The sequence was performed according to the protocol in Wang et al 2013, or “MC1R Canine Sequencing paper” on Moodle and also performed PCR on our certain traits or SNPs that we chose (herding). Then, they were sent off for sequencing and ExoSAP-It PCR Product Cleanup was used to remove the primers before the sequencing.
6. Fifth Lab of Canine Experiment - DNA Analysis
This lab we followed the protocol from the “Analysis of Canine DNA Sequencing” file on Moodle. We used “4Peaks” to clean up our files of the sequencing of the L and M dog samples. Then we assembled and uploaded the overlapping sequencing to “CAP3 Sequence Assembly” program with the FASTA format. After that, we uploaded our contig to Moodle and shared them with the class. After, we analysed and compared our sequences on “NCBI” by doing a Nucleotide Blast. Then, we aligned the class’ data. There were several problems with this step due to incorrect formatting. After, we compared the canine M1 and L2 sequences with mammoths, neanderthals, horses, and cats with the “Muscle” program