The study for the phantom limb sites served a micro-niche of patients (amputated limb, phantom pain, willing participation), but the experimenters were able to find participants with amputated limbs that were willing to perform in the study. The study was opportunistic and the experimenters took advantage of the resources they had at the time. Using humans rather than animals served an advantage in the specificity of the data they obtained. The study was meant to not only lead to better knowledge of the thalamus, it also allowed them to pinpoint the source of the phantom limb pain and help the patients that had it.
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Since the study served such a micro-niche of patients (amputated limb, phantom pain, willing participation), it is incredible that they were able to find participants with amputated limbs that were willing to perform in the study. The study was very opportunistic and the experimenters took advantage of the resources they had at the time. Also using humans rather than animals served an advantage in the specificity of the data they obtained. The study was meant to not only led to better knowledge of the thalamus, it also allowed them to pinpoint the source and help the patients that have phantom limb pain.
Some of the weaknesses of this study include the small sample size of patients, the variability in amputated limbs such as the arm and the leg, and the different times of amputation. Some of the amputees had their arm or leg amputated much more recently than others. All of these variables could lead to significant differences in their results. The results however, do seem valid since the results showed that the patients with phantom limb pain had areas in their thalamus that remained active while those that didn’t feel anything had that area rearranged; the results showed a general trend among all of the patients.
The experimenters showed that in patients with phantom sensations, there is a thalamic representation of that missing limb that continues to be active thus causing the sensations and pain felt in the missing limb. Another conclusion is that the loss of the limb caused the thalamic representation of the stump to expand (in some patients more than others). Yes, the conclusions follow logically from the design of the experiment and the results. They tested for receptive fields and then looked at the projected fields and noticed mismatches.
For the purification of the extracted dog DNA, the Nanodrop indicated that the DNA concentration was 29.6 nanograms/ microliter and that the 260/280 ratio was 1.77.
The gel electrophoresis of the MC1R PCR of the dog DNA for JP displayed on Well 2 showed that it had a band near 1 kb which was similar to the other samples of dog DNA from other groups. The band for JP appeared horizontally straight with no curvatures (Fig. 1).
In the analysis of the DNA sequence of JP, there was a single nucleotide polymorphism of 790 A > G. After the BLAST, the first top hit was Canis lupus familiaris boxer breed chromosome 5 which was 99% identical to the contiguous sequence. Also, 16A and 16C did not show any heterozygous bases while 16B showed one at C402: C/T.
In the analysis of the protein structure of JP, there was a mismatch between the reference sequence of amino acids and the sequence for JP at p.Met264Val.
A restriction digest was performed on the dCAPS PCR products to observe the differences in the two alleles of each SNP. The methods from “Restriction digest of dCAPS PCR products” were followed for this section. The restriction digest conditions were researched on the NEB website and the amount of each ingredient for the digest were calculated. It was determined that for DS12, the enzyme HpaII would be used with 1xCutsmart buffer while for DS1S, the enzyme BamHI would be used with NE buffer 3. Each needed an incubation time of 1 hour at 37°C. A chain of eight PCR tubes were provided. Four would be cut and four would be uncut mixes. For the uncut mixes, 10 microliters of PCR product and 10 microliters of water were added. For the cut mixes, 10 microliters of PCR product and 7 microliters of water were added. Instead of the intended 2 microliters of buffer, 20 microliters of buffer were added to each of the regular mixes accidentally. With no remaining PCR product, a new batch was not available and so the 1 microliter of restriction enzyme was not added since the ratio would restrict the function of the reaction anyway. The digests were incubated for 1 hour. After incubation, 12 microliters of each sample was mixed with 3 microliters of loading dye on a Parafilm sheet and loaded into separate columns of a gel for gel electrophoresis (Loomis 2017).
Derived cleaved amplified polymorphic sequences (dCAPS) were performed to identify the genotype of the SNP that is highly associated with the trait for Earbend (CFA34, DS12). The procedure was taken from “dCAPS PCR Primer Design Protocol.” All of the SNP and primer information were recorded onto a spreadsheet. The dCAPS primer was designed by using a website that searches for a compatible restriction endonuclease to digest the specific amplified product. The wildtype and mutant SNP sequences were formatted and added to the site. The number of mismatches for primers were determined to limit the different potential enzymes to ones more readily available; there were two mismatches before finding an available enzyme. The enzyme HpaII with the recognition site CCGG was chosen. A second primer was found using the Primer3 program and entering the full sequence and the forward primer. The reverse primer was found and the size of the digested PCR product was calculated. A positive control primer was designed using the forward primer (Loomis 2017).
The MC1R DNA sequences were analyzed and cleaned by cropping areas that were not as reliable such as the 5’ and 3’ ends which contained poorer quality sequences than the middle sections. This part of the procedure was done following “Analysis of Canine DNA Sequencing.” Heterozygous sequences were noted for each sequence. The three files were refined and assorted into FASTA format. The overlapping sequences were assembled into one contiguous sequence using the CAP3 Sequence Assembly Program. The contiguous sequence was then compared to known sequences on the NCBI database using BLAST. Sequence matches were recorded and reviewed for potential missense SNPs. The sequence was compared to a consensus sequence on NCBI and aligned using the EMBOSS Needle Pairwise Sequence Alignment program. The SNPs were identified and recorded.
Using the DNA extracted, the MC1R gene was amplified using a Polymerase Chain Reaction. The procedure for performing this section was from “Polymerase Chain Reaction.” The master mix for the PCR was created by adding water, PCR buffer 10x, dNTP mix, forward primer, reverse primer, and Taq polymerase; enough master mix was made for five samples (one extra sample). Two identical PCR samples were made for each group. Each PCR sample consisted of 10 microliters of master mix, 3.38 microliters of 100 ng DNA, and 6.62 microliters of water which were added to a PCR tube. The PCR strip was placed in the thermocycler for amplification.
The next step was to take the MC1R PCR products and perform a gel electrophoresis. The procedure was followed by using “Sample Preparation for Sequencing” The two identical samples were combined, and 12 microliters was added to a sheet of parafilm in addition to 2 microliters of loading dye. To make a point of reference, 7 microliters of DNA ladder was added to one of the columns. The DNA mixture was added to the second column and the gel was run after all the other samples were loaded. The remaining PCR product was then purified from any residual primers using ExoSAP-IT. Three separate tubes were used; 7 microliters of PCR product and 3 microliters of ExoSAP-IT were added to each tube and the tubes were incubated. After incubation, the samples were sent for DNA sequencing.
The dog DNA was extracted from the cells and purified using three steps: lysing the cells, forming the DNA in the QIAamp spin column and thoroughly washing it, and eluting the DNA and moving it to a collection tube. The procedures were performed by following “Genomic DNA purification from buccal swab using the QIAamp DNA Mini Kit.” For lysing the cells, Buffer AL was added, the mixture was vortexed, incubated, and then vortexed again. For precipitating the DNA into the column, 100% ethanol was added to the mixture and then centrifuged. Some of the mixture was then added to the column and then centrifuged and the tube was emptied. This step was repeated to increase the concentration of DNA in the column. For washing the DNA, two alcoholic solutions were added. Buffer AW1 was added first, centrifuged, and emptied; then Buffer AW2 was added, centrifuged, and emptied. Elution and collection of the DNA consisted of adding Buffer AE, incubating, and centrifuging the sample. The solution was disposed of accidentally; since the DNA was in the collection tube, the elution steps had to be performed again. The steps for elution were repeated for a second time to produce a higher yield of DNA.