Water Foul

The purpose of this experiment was to utilize polymerase chain reaction and agarose gel electrophoresis to identify, amplify, and compare a conserved region of the DRD4 gene across six orders within the Class Mammalia. It was hypothesized that if primers were designed for a region of DNA with an 85% similar sequence of about 20 nucleotides, then the primers would anneal correctly to yield the proper conserved region of the DRD4 gene, because designed primers need to have a series of four or more nucleotides at the 3' end of the primer to bind properly to the complementary base to allow Taq polymerase to elongate the DNA copies from the 3' end. Throughout the trials, both the human and homolog DNA produced bands that ran below the dye front ranging from 70 base-pairs to 412 base-pairs. All of these were within reasonable range of the anticipated base pair amounts; however, not enough trials were run to conclude conservation of the DRD4 gene across the Class Mammalia. A secondary set of bands was observed within the Order Artiodactyla ranging from 1,200 base-pairs to 2,293 base-pairs. This will need to be researched further to determine the source of the outlier bands. If further research and trials were conducted for all orders, the conservation could contribute to the knowledge of the DRD4 gene and risk-taking behaviors throughout mammals. By understanding this, novelty seeking behaviors could be discovered in more animals, which would allow for a wider scope of animal models for medical research advances, such as ADHD which has been linked to the DRD4 gene (Wang et al, 2004).

Figure 1 - The DRD4 Gene Across Six Different Orders Within the Class Mammalia. (A) The Phylogenetic Tree That Illustrates the Common Ancestry of The Animals That Were Studied. The phylogentic tree includes humans (Homo sapiens), chinchilla (Chinchilla lanigera), rabbit (Oryctolagus cuniculus), skunk (Mephitis mephitis), dog (Canis lupus familiaris), donkey (Equus asinus), horse (Equus caballus), llama (Lama glama), pig (Sus scrofa domesticus), goat (Capra hircus), and cow (Bos taurus). (B) Animals from the Class Mammalia to Construct Primers. Animals from multiple orders under the Class Mammalia were run through an online program called the National Center for Biotechnology Information BLAST, or NCBI BLAST (Bethesda, Maryland). This is a system that will align and match similar genome sequences of all organisms. When the DRD4 gene was run through the program, a multitude of animals were matched to the human genome. Animals of the following orders were matched: Order Primates, Order Lagomorpha, Order Rodentia, Order Carnivora, Order Artiodactyla, and Order Perissodactyla. A select number of these animals were run through the machine a second time in order to get a proper alignment. A portion of the alignment is shown with human (Order Primates), horse (Order Perissodactyla), dog (Order Carnivora), and chinchilla (Order Rodentia). The section shown in yellow are the areas that were used to design the primers. The letters shown in red are a specific nucleotide location that does not match up within the animals shown, such as human, horse, dog, and chinchilla. The stars that are above the highlighted primer section indicate the sections in the primer that were designed as a complementary base-pair that can match up with adenine, thymine, guanine, and cytosine. The forward primer started at the human base-pair location of 2,165 and ended at human base-pair location of 2,184. The forward primer was as follows: 5'-TGCGACGIICTCATGGCIAT-3'. The reverse primer started at the human base-pair location 2,501 and ended at human base-pair location of 2,520. The reverse primer was as follows: 5'-GCGGAICTIITGGCGCTGAT-3'.

Figure 2-Epoch Spectrophotometer DNA Sample Readings.(A)Three Human DNA Samples Tested for DNA Quantity. The three human samples were also run through an Epoch Spectrophotometer with Gen5 Software. Once the blanks were read with double distilled H₂O, to calibrate the machine, the samples were loaded respectively onto the Take-3 Plate. Each of the samples were read for a concentration and total DNA amounts were calculated. Each sample was 0.3 mL. Human DNA sample 1 had a concentration of 31.0 ng/µL and a total DNA quantity of 9,300 ng. Human DNA sample 2 had a concentration of 18.0 ng/µL and a total DNA quantity of 5,400 ng. Human DNA sample 3 had a concentration of 8.0 ng/µL and a total DNA quantity of 2,400 ng. Due to this information, DNA sample 1 was used for further DNA experimentation for humans. (B) Ten Animal DNA Samples Tested for DNA Quantity.Ten animal samples were run through an Epoch Spectrophotometer with Gen5 Software. Once the blanks were read with double distilled H₂O, to calibrate the machine, the samples were loaded respectively onto the Take-3 Plate. Each of the samples were read for a concentration and total DNA amounts were calculated. Each sample was 1.25 mL. The skunk DNA sample had a concentration of 3,341.5 ng/µL and a total DNA quantity of approximately 4,176,875 ng. The horse DNA sample had a concentration of 3,274.5 ng/µL and a total DNA quantity of approximately 4,093,125 ng. The pig DNA sample had a concentration of 3,354.0 ng/µL and a total DNA quantity of approximately 4,192,500 ng. The llama DNA sample had a concentration of 2,950.5 ng/µL and a total DNA quantity of approximately 3,688,125 ng. The dog DNA sample had a concentration of 3,290 ng/µL and a total DNA quantity of approximately 4,112,500 ng. The donkey DNA sample had a concentration of approximately 3,384 ng/µL and a total DNA quantity of approximately 4,230,000 ng. The goat DNA sample had a concentration of 3,294.0 ng/µL and a total DNA quantity of approximately 4,117,500 ng. The cow DNA sample had a concentration of 3,143.5 ng/µL and a total DNA quantity of approximately 3,929,375 ng. The rabbit DNA sample had a concentration of 3,350.5 ng/µL and a total DNA quantity of approximately 4,188,125 ng. The chinchilla DNA sample had a concentration of 3,266.5 ng/µL and a total DNA quantity of approximately 4,083,125 ng.

Figure 3 - Conserved Region of DRD4 Gene Within the Order Primates. The forward primer was 5'-TGCGACGIICTCATGGCIAT-3'. The reverse primer that was 5'-GCGGAICTIITGGCGCTGAT-3'.The entire length of the conserved region of the DRD4 gene for humans was 355 base-pairs.One cocktail was created for the Order Primates. The cocktail was made with 1.0 µL 100.0 µM Designed Forward Primer, 1.0 µL 100.0 µM Designed Reverse Primer, 1.0 µL 5U/µL Taq Polymerase, 7.0 µL 10 X PCR Buffer + MgCl₂ (20.0 mM Tris-HCl, 10.0 mM (NH₄)₂SO₄, 10.0 mM KCl, 2.0 mM MgSO₄, 0.1% Triton^® X-100, pH 8.8), 1.0 µL 0.5 mM dNTPs, and 38.0 µL Nuclease-Free H₂O. 1.0 µL of the human #1 DNA suspension was added to the cocktail. For our positive control, 1.0 µL of the human #1 DNA suspension was substituted with 1.0 µL of Lambda Rz Virus DNA, and the respective primers were substituted. The DNA suspension that was run through PCR with the #144901 and #146840 primers was also used as a positive control for the designed primers being tested on humans and the homolog species. For our negative control, the 1.0 µL of the human #1 DNA suspension was replaced with 1.0 µL Nuclease-Free H₂O. The PCR cocktails were run through the Bio-Rad T-100 Thermal Cycler, except for the negative control cocktail. The initial cycle was performed at 95°C for three minutes. The cocktails were ran through 35 cycles of 95°C for 30 seconds, 49°C for 30 seconds, and 72°C for one minute. An extension cycle for two minutes at 72°C was also used. A 0.8% agarose gel was made with 1 X TBE (Tris/Borate/EDTA) buffer, agarose powder, and SyBr Safe dye. It was run at 135 V for 20 minutes. Lane 1 was loaded with 7.0 µL of Kb Plus Ladder and 3.0 µL of 6X Gel Loading Dye. Lane 2 was loaded with 7.0 µL of the negative control and 3.0 µL of 6X Gel Loading Dye. Lane 3 was loaded with 7.0 µL of the Lambda DNA cocktail and 3.0 µL of 6X Gel Loading Dye. Lane 4 was loaded with 7.0 µL of Human DNA cocktail made with primer #144901 and #146840 and 3.0 µL of 6X Gel Loading Dye. Lane 6 was loaded with 7.0 µL of Human #1 DNA cocktail and 3.0 µL of 6X Gel Loading Dye. (A) Trial One Gel for the Order Primates with the Semi-log Graph. In Lane 3, the positive control using Lambda Rz Virus traveled 4.2672 cm from the well and was calculated to be approximately 345 base-pairs in length. In Lane 4, the positive control with primer #144901 and #146840 traveled 4.5212 cm and was 241 base-pairs(Wang et al, 2004). In Lane 5, the Human #1 DNA traveled 4.9784 cm and was 127 base-pairs. (B) Trial Two Gel for the Order Primates with the Semi-Log Graph. The same methods were applied from part (A) above. In Lane 3, the positive control using Lambda Rz Virus traveled 2.8956 cm and was 656 base-pairs. In Lane 4, the positive control with primer #144901 and #146840 traveled 3.1242 cm and was 520 base-pairs (Wang et al, 2004). In Lane 5, the Human #1 DNA traveled 3.3528 cm and 412 base-pairs.

Figure 4 - Conserved Region of DRD4 Gene Within the Order Carnivora. The forward primer was 5'-TGCGACGIICTCATGGCIAT-3'. The reverse primer was 5'-GCGGAICTIITGGCGCTGAT-3'. The entire length of the conserved region of the DRD4 gene for dogs was 254 base-pairs. The skunk's conserved region of the DRD4 gene was expected to be approximately 254 base-pairs because it is so closely related to the dog. The cocktails were made with 1.0 µL 100.0 µM Designed Forward Primer, 1.0 µL 100.0 µM Designed Reverse Primer, 1.0 µL 5U/µL Taq Polymerase, 7.0 µL 10 X PCR Buffer + MgCl₂ (20.0 mM Tris-HCl, 10.0 mM (NH₄)₂SO₄, 10.0 mM KCl, 2.0 mM MgSO₄, 0.1% Triton^® X-100, pH 8.8), 1.0 µL 0.5 mM dNTPs, and 38.0 µL Nuclease-Free H₂O. 1.0 µL of the dog DNA suspension was added to one cocktail and added 1.0 µL of the skunk DNA suspension to a second cocktail. For our positive control, animal DNA was substituted with 1.0 µL of Lambda Rz Virus DNA with the respective primers. The DNA suspension that was run through PCR with #144901 and #146840 primers was also used as a positive control. For our negative control, DNA was replaced with 1.0 µL Nuclease-Free H₂O. The PCR cocktails were run through the Bio-Rad T-100 Thermal Cycler, except for the negative control cocktail. The initial cycle was performed at 95°C for three minutes. The cocktails were ran through 35 cycles of 95°C for 30 seconds, 49°C for 30 seconds, and 72°C for one minute. An extension cycle for two minutes at 72°C was also used. A 0.8% agarose gel was made with 1 X TBE (Tris/Borate/EDTA) buffer, agarose powder, and SyBr Safe dye. It was run at 135 V for 20 minutes. Lane 1 was loaded with 7.0 µL of Kb Plus Ladder and 3.0 µL of 6X Gel Loading Dye. Lane 2 was loaded with 7.0 µL of the negative control and 3.0 µL of 6X Gel Loading Dye. Lane 3 was loaded with 7.0 µL of the Lambda DNA cocktail and 3.0 µL of 6X Gel Loading Dye. Lane 4 was loaded with 7.0 µL of Human DNA cocktail made with primer #144901 and #146840 and 3.0 µL of 6X Gel Loading Dye. Lane 5 was loaded with 7.0 µL of Skunk DNA cocktail and 3.0 µL of 6X Gel Loading Dye. Lane 6 was loaded with 7.0 µL of Dog DNA cocktail and 3.0 µL of 6X Gel Loading Dye. (A) Trial One Gel for the Order Carnivora with the Semi-log Graph. There were no bands produced for the positive control Lambda Rz virus in Lane 3, the positive control with primer #144901 and #146840 in Lane 4, dog PCR cocktail in Lane 5, and skunk PCR cocktail in Lane 6 (Wang et al, 2004). (B) Trial Two Gel for the Order Carnivora with the Semi-Log Graph. In Lane 3, the positive control using Lambda Rz Virus traveled 3.4798 cm and was 345 base-pairs. In Lane 4, the positive control with primer #144901 and #146840 traveled 4.1148 cm and was 163 base-pairs (Wang et al, 2004). In Lane 5, the dog DNA traveled 4.2418 cm and was 141 base-pairs. In Lane 6, the skunk DNA traveled 4.1656 cm and was 154 base-pairs.

Figure 5 - Conserved Region of DRD4 Gene Within the Order Artiodactyla. The forward primer was 5'-TGCGACGIICTCATGGCIAT-3'. The reverse primer was 5'-GCGGAICTIITGGCGCTGAT-3'. The conserved region of the DRD4 gene was expected to be 250-350 base-pairs for all of Artiodactyla, due to relation to Carnivora and Perissodactyla. The cocktails were 1.0 µL 100.0 µM Designed Forward Primer, 1.0 µL 100.0 µM Designed Reverse Primer, 1.0 µL 5U/µL Taq Polymerase, 7.0 µL 10 X PCR Buffer + MgCl₂ (20.0 mM Tris-HCl, 10.0 mM (NH₄)₂SO₄, 10.0 mM KCl, 2.0 mM MgSO4, 0.1% Triton^® X-100, pH 8.8), 1.0 µL 0.5 mM dNTPs, and 38.0 µL Nuclease-Free H₂O. 1.0 µL of the each DNA suspension was added to its own cocktail. For a positive control, DNA was replaced with 1.0 µL of Lambda with its own primers.The DNA cocktail with #144901 and #146840 primers was a positive control. DNA was replaced with 1.0 µL Nuclease-Free H₂O as a negative control. The PCR cocktails were run through the Bio-Rad T-100 Thermal Cycler, except for negative control. The initial cycle was run at 95°C for three minutes. They were ran through 35 cycles of 95°C for 30 seconds, 49°C for 30 seconds, and 72°C for one minute. An extension cycle for two minutes at 72°C used. A 0.8% gel was made with 1 X TBE buffer, agarose, and SyBr Safe. It was run at 135 V for 20 minutes. Lane 1 was loaded with 7.0 µL of Kb Plus Ladder and 3.0 µL of 6X Loading Dye. Lane 2 was loaded with 7.0 µL of the negative control and 3.0 µL of 6X Loading Dye. Lane 3 was loaded with 7.0 µL of the Lambda cocktail and 3.0 µL of 6X Loading Dye. Lane 4 was loaded with 7.0 µL of DNA cocktail with primer #144901 and #146840 and 3.0 µL of 6X Loading Dye. Lane 5 through Lane 8 were loaded with 7.0 µL of animal DNA cocktails and 3.0 µL of 6X Loading Dye. (A) Trial One Gel for the Order Artiodactyla with the Semi-log Graph. In Lane 3, the Lambda moved 3.7084 cm and was 725 base-pairs. In Lane 4, the primer #144901 and #146840 cocktail moved 4.7752 cm and was 160 base-pairs (Wang et al, 2004). In Lane 5, the cow DNA moved 4.5974 cm and was 206 base-pairs (solid), and it moved 3.175 cm and was 1,544 base-pairs (dashed). In Lane 6, the goat DNA moved 4.572 cm and was 213 base-pairs (solid), and it moved 2.8956 cm and was 2,293 base-pairs (dashed). In Lane 7, the pig DNA moved 4.5466 cm and was 221 base-pairs (solid), and it moved 3.3528 cm and was 1,200 base-pairs (dashed). In Lane 8, the llama DNA moved 4.4958 cm and was 238 base-pairs (solid), and it moved 3.175 cm and was 1,544 base-pairs (dashed). (B) Trial Two Gel for the Order Artiodactyla with the Semi-Log Graph. In Lane 3, the Lambda moved 4.318 cm and was 439 base-pairs. In Lane 4, the cocktail with primer #144901 and #146840 moved 5.715 cm and was 75 base-pairs (Wang et al, 2004). In Lane 5, the cow DNA moved 5.7658 cm and was 70 base-pairs. In Lane 6, the goat DNA moved 5.588 cm and was 88 base-pairs. In Lane 7, the pig DNA moved 5.5118 cm and was 97 base-pairs. In Lane 8, the llama DNA moved 5.4356 cm and was 107 base-pairs.

Figure 6 - Conserved Region of DRD4 Gene Within the Order Perissodactyla. The forward primer was 5'-TGCGACGIICTCATGGCIAT-3'. The reverse primer was 5'-GCGGAICTIITGGCGCTGAT-3'. The length of the conserved region of the DRD4 gene for horse was 344 base-pairs.The donkey's region of the DRD4 gene was expected to be 344 base-pairs, because of the relation. The cocktails were 1.0 µL 100.0 µM Designed Forward Primer, 1.0 µL 100.0 µM Designed Reverse Primer, 1.0 µL 5U/µL Taq Polymerase, 7.0 µL 10 X PCR Buffer + MgCl₂ (20.0 mM Tris-HCl, 10.0 mM (NH₄)₂SO₄, 10.0 mM KCl, 2.0 mM MgSO₄, 0.1% Triton^® X-100, pH 8.8), 1.0 µL 0.5 mM dNTPs, and 38.0 µL Nuclease-Free H₂O. For a positive control, DNA was substituted with 1.0 µL of Lambda DNA with its primers. The DNA suspension that was run through PCR with #144901 and #146840 primers was a positive control. For the negative control, DNA was replaced with an additional 1.0 µL Nuclease-Free H₂O. The PCR cocktails were run through the Bio-Rad T-100 Thermal Cycler, except for the negative control cocktail. The initial cycle was performed at 95°C for three minutes. The cocktails were ran through 35 cycles of 95°C for 30 seconds, 49°C for 30 seconds, and 72°Cfor one minute. An extension cycle for two minutes at 72°C was also used. A 0.8% agarose gel was made with 1 X TBE buffer, agarose powder, and SyBr Safe dye. It was run at 135 V for 20 minutes. Lane 1 was loaded with 7.0 µL of Kb Plus Ladder and 3.0 µL of 6X Loading Dye. Lane 2 was loaded with 7.0 µL of the negative control and 3.0 µL of 6X Loading Dye. Lane 3 was loaded with 7.0 µL of the Lambda DNA cocktail and 3.0 µL of 6X Loading Dye. Lane 4 was loaded with 7.0 µL of cocktail made with primer #144901 and #146840 and 3.0 µL of 6X Loading Dye. Lane 5 was loaded with 7.0 µL of horse DNA cocktail and 3.0 µL of 6X Loading Dye. Lane 6 was loaded with 7.0 µL of donkey DNA cocktail and 3.0 µL of 6X Loading Dye. (A) Trial One Gel for the Order Perissodactyla with the Semi-log Graph. In Lane 3, the Lambda moved 4.4704 cm and was 392 base-pairs. In Lane 4, the cocktail with primer #144901 and #146840 moved 5.6134 cm and was 142 base-pairs (Wang et al, 2004). In Lane 5, the horse DNA moved 5.6896 cm and was 132 base-pairs. In Lane 6, the donkey DNA moved 5.7404 cm and was 127 base-pairs. (B) Trial Two Gel for the Order Perissodactyla with the Semi-Log Graph. In Lane 3, Lambda moved 3.2766 cm and was 484 base-pairs. In Lane 4, the positive control with primer #144901 and #146840 moved 4.0894 cm and was 187 base-pairs (Wang et al, 2004). In Lane 5, the horse DNA moved 3.937 cm and was 223 base-pairs. In Lane 6, the donkey DNA moved 3.9878 cm and was 211 base-pairs.

Figure 7 - Conserved Region of DRD4 Gene Within the Order Rodentia. The forward primer was 5'-TGCGACGIICTCATGGCIAT-3'. The reverse primer was 5'-GCGGAICTIITGGCGCTGAT-3'.The entire length of the conserved region of the DRD4 gene for chinchilla was 253 base-pairs. The cocktail was made with 1.0 µL 100.0 µM Designed Forward Primer, 1.0 µL 100.0 µM Designed Reverse Primer, 1.0 µL 5U/µL Taq Polymerase, 7.0 µL 10 X PCR Buffer + MgCl₂ (20.0 mM Tris-HCl, 10.0 mM (NH₄)₂SO₄, 10.0 mM KCl, 2.0 mM MgSO₄, 0.1% Triton ^®X-100, pH 8.8), 1.0 µL 0.5 mM dNTPs, and 38.0 µL Nuclease-Free H₂O. 1.0 µL of the chinchilla DNA suspension was added to the cocktail. For the positive control, 1.0 µL of the chinchilla DNA suspension was substituted with 1.0 µL of Lambda, and the respective primers were substituted. The DNA suspension that was run through PCR with #144901 and #146840 primers was also used as a positive control for the designed primers being tested on humans and the homolog species. For the negative control, the 1.0 µL of the chinchilla DNA suspension was replaced with an additional 1.0 µL Nuclease-Free H₂O. The PCR cocktails was run through the Bio-Rad T-100 Thermal Cycler, except for the negative control cocktail. The initial cycle was performed at 95°C for three minutes. The cocktails were ran through 35 cycles of 95°C for 30 seconds, 49°C for 30 seconds, and 72°C for one minute. An extension cycle for two minutes at 72°C was also used. A 0.8% agarose gel was made with 1 X TBE buffer, agarose, and SyBr Safe and was run at 135 V for 20 minutes. Lane 1 was loaded with 7.0 µL of Kb Plus Ladder and 3.0 µL of 6X Loading Dye. Lane 2 was loaded with 7.0 µL of the negative control and 3.0 µL of 6X Loading Dye. Lane 3 was loaded with 7.0 µL of the Lambda DNA cocktail and 3.0 &micoL of 6X Loading Dye. Lane 4 was loaded with 7.0 µL of Human DNA cocktail made with primer #144901 and #146840 and 3.0 µL of 6X Loading Dye. Lane 5 was loaded with 7.0 µL of Chinchilla DNA cocktail and 3.0 µL of 6X Loading Dye. (A) Trial One Gel for the Order Rodentia with the Semi-log Graph. In Lane 3, the positive control using Lambda moved 3.7592 cm and was 273 base-pairs. In Lane 4, the positive control with primer #144901 and #146840 did not appear in the gel (Wang et al, 2004). In Lane 5, the chinchilla DNA moved 4.3688 cm and was 130 base-pairs. (B) Trial Two Gel for the Order Rodentia with the Semi-Log Graph. The same methods were applied from part (A) above. In Lane 3, the positive control Lambda moved 3.9116 cm and was 424 base-pairs. In Lane 4, the positive control with primer #144901 and #146840 moved 4.4704 cm and was 187 base-pairs (Wang et al, 2004). In Lane 5, the chinchilla DNA moved 4.4196 cm and was 202 base-pairs.

Figure 8 - Conserved Region of DRD4 Gene Within the Order Lagomorpha. The forward primer was 5'-TGCGACGIICTCATGGCIAT-3'. The reverse primer was 5'-GCGGAICTIITGGCGCTGAT-3'. The entire length of the conserved region of the DRD4 gene for chinchilla was 253 base-pairs. The rabbit's conserved region of the DRD4 gene was expected to be 253 base-pairs since rabbits and chinchilla are closely related. The cocktails were 1.0 µL 100.0 µM Designed Forward Primer, 1.0 µL 100.0 µM Designed Reverse Primer, 1.0 µL 5U/µL Taq Polymerase, 7.0 µL 10 X PCR Buffer + MgCl₂ (20.0 mM Tris-HCl, 10.0 mM (NH₄)₂SO₄, 10.0 mM KCl, 2.0 mM MgSO₄, 0.1% Triton ^®X-100, pH 8.8), 1.0 µL 0.5 mM dNTPs, and 38.0 µL Nuclease-Free H>sub>2O. 1.0 µL of the rabbit DNA suspension to the cocktail. For the positive control, DNA was substituted with 1.0 µL of Lambda DNA with its own primers.. The cocktail that was run through PCR with #144901 and #146840 primers was a positive control. For the negative control, the DNA was replaced with 1.0 µL Nuclease-Free H₂O. The PCR cocktails was run through the Bio-Rad T-100 Thermal Cycler, except for the negative control cocktail. The initial cycle was performed at 95°C for three minutes. The cocktails were ran through 35 cycles of 95°C for 30 seconds, 49°C for 30 seconds, and 72°C for one minute. An extension cycle for two minutes at 72°C was also used. A 0.8% gel was made with 1 X TBE buffer, agarose powder, and SyBr Safe dye and was run at 135 V for 20 minutes. All of the samples were mixed with loading dye before being loaded into the gel. Lane 1 was loaded with 7.0 µL of Kb Plus Ladder and 3.0 µL of 6X Loading Dye. Lane 2 was loaded with 7.0 µL of the negative control and 3.0 µL of 6X Loading Dye. Lane 3 was loaded with 7.0 µL of the Lambda DNA cocktail and 3.0 µL of 6X Loading Dye. Lane 4 was loaded with 7.0 µL of Human DNA cocktail made with primer #144901 and #146840 and 3.0 µL of 6X Loading Dye. Lane 5 was loaded with 7.0 µL of Rabbit DNA cocktail and 3.0 µL of 6X Loading Dye. (A) Trial One Gel for the Order Lagomorpha with the Semi-log Graph. In Lane 3, the Lambda traveled 4.445 cm and was 351 base-pairs. In Lane 4, the positive control with primer #144901 and #146840 traveled 4.8006 cm and was 230 base-pairs (Wang et al, 2004). In Lane 5, the rabbit DNA traveled 5.1308 cm and was 156 base-pairs. (B) Trial Two Gel for the Order Lagomorpha with the Semi-Log Graph. In Lane 3, the Lambda traveled 4.064 cm and was 325 base-pairs. In Lane 4, the positive control with primer #144901 and #146840 moved 5.08 cm and was 93 base-pairs (Wang et al, 2004). In Lane 5, the rabbit DNA moved 5.0546 cm and was 96 base-pairs.

The purpose was to identify, amplify, and compare conserved regions of the DRD4 gene across six orders within the Class Mammalia. It was hypothesized that if primers are designed for a region of DNA with an 85% similar sequence of about 20 nucleotides, then the primers will anneal correctly to yield the proper conserved region of the DRD4 gene, because designed primers need to have a series of four or more nucleotides at the 3' end of the primer bind properly to the complementary base to allow Taq polymerase to elongate the DNA copies from the 3' end.

Novelty seeking behaviors are behaviors that are considered exploratory. These consist of diminished harm avoidance in the presence of humans in animals or drug abuse in humans (Mallard et al, 2016). The Dopamine Receptor D4 gene, or the DRD4 gene has been linked to novelty seeking behaviors through a positive reinforcement loop (Paterson et al, 1999; Ebstein et al, 1997). Dopamine is a neurotransmitter that binds to the D4 receptor to control reward, pleasure, and positive reinforcement signals throughout the brain and body (Ebstein et al, 1997). Dopamine is released in response to reward or pleasure; this causes the signals to be activated in response to those external cues (Ebstein et al, 1997). The link between novelty seeking behavior and the DRD4 gene throughout the Class Mammalia could lead to a multitude of animal models for medical research testing in humans relating to novelty seeking behaviors and diseases that have an etiology based within the DRD4 gene.

The methods for experimentation included the following: DNA extraction, DNA preparation and purification, polymerase chain reaction (PCR), and agarose gel electrophoresis. Controls were established to ensure that the data observed was valid and reliable. A negative control was created by substituting DNA in the PCR cocktails with an additional 1.0 µL of Nuclease-Free H₂O. This cocktail was not run through PCR. By including all other PCR ingredients other than the DNA, the negative control proved that the DNA was the component generating the bands that were appearing within the agarose gels. One positive control that was utilized was the Lambda Rz Virus. The same PCR cocktails and PCR protocols were employed in the experimental cocktails and the Lambda cocktails. This positive control proved that the PCR cocktail and PCR protocols were fully functional and should have worked for the experimental cocktails. Another positive control that was implemented was the human DNA sample cocktail that was run through PCR using primer #144901 and primer #146840 (Wang et al, 2004). This positive control established that the PCR cocktails and PCR protocols that were being conducted were not only working for prokaryotic DNA, but also for eukaryotic DNA, thus creating the DNA bands within the agarose gel. Although many controls were set in place, there were some weaknesses within the protocol of the experiment. One weakness was that the positive control with previously studied primers proved that the PCR protocol would work for eukaryotic organisms in general, but the control did not prove that it would work for the specific region of conservation being tested for since the primers were for a different region of the DRD4 gene (Wang et al, 2004). Due to the lack of previous research on the region of conservation being tested, this was the most specific control that could be employed during experimentation. Another weakness was that the negative control did not undergo PCR. The negative control proved that the DNA was creating the bands; however, the negative control did not account for other elements being able to generate bands within the agarose gel after being run through PCR without DNA being added to the cocktail.

It was predicted that the human DNA would create a band that was approximately 355 base-pairs in length because the forward primer started at base-pair 2,165 and ended at base-pair 2,520 as determined from NCBI BLAST (Bethesda, Maryland). It was predicted that the horse DNA would create a band that was approximately 344 base-pairs in length, the dog DNA would create a band that was approximately 254 base-pairs in length, and the chinchilla DNA would create a band that was approximately 253 base-pairs in length because the forward primer started at base-pair 2,165 and ended at 2,520 with gaps within the conserved region as determined from NCBI BLAST (Bethesda, Maryland). After two trials for each order within the Class Mammalia, the bands generated within the agarose gels ranged from 70 base-pairs to 412 base-pairs. Although those numbers are not as close to the exact base-pair amounts that were calculated, the length of conserved region of the DRD4 gene may have had some variability across the different orders. That being said, the data that was gathered was inconclusive due to lack of trials and concrete evidence that pointed to a more marked conservation. In order to support a solid finding, further research would have to be conducted. Even though the bands that were generated were within reasonable range to the predictions, there were a couple irregularities within the agarose gels. One irregularity is the secondary region of bands that became prominent within the Order Artiodactyla. The order had a region of bands that ranged from 1,200 base-pairs to 2,293 base-pairs. This was a larger region that had been amplified that was unexpected. Due to the unknown origin of this location of bands, further research would have to be performed to determine the causality. Another irregularity was the lack of a band for the Lambda Rz Virus positive control within random agarose gels. This could have been caused by a lack of a reagent of the PCR cocktail in one of the multiple cocktails that were prepared for the Lambda positive control. Further research will have to be conducted to remove all or most of the irregularities that occurred within the agarose gels.

Ten animal fecal samples were obtained from Green Acres Farm (Highland, Michigan) in early February 2017. Each sample was preserved until use in early April 2017. The homolog samples exhibited high DNA amounts within the suspension; however, they still produced very faint bands. For further research, experiments would have been run within 48 hours of sample collection. This would clarify if the DNA was degraded from the age of the sample.

For further experimentation on the DRD4 gene, a wider expanse of animals from a larger sample of orders within the Class Mammalia would have been studied. The Order Proboscidea would have been added with African Elephant (Loxodonta Africana). The Order Tubulidentata would have been added with Aardvark (Orycteropus afer afer). The Order Marsupialia would have been added with Koala (Phascolarctos cinereus). The Order Cetacea would have been added with Common Dolphin (Delphinus delphis), Humpback Dolphin (Sousa delphis), and Bottlenose Dolphin (Tursiops australis). More mammals being studied would further extend the phylogenetic tree and would provide a clearer understanding of the conserved region of the DRD4 gene across multiple orders within the Class Mammalia.