Written by: B200
Revised by: B315
Finalized by: B180
The Monoamine Oxidase A (MAOA) gene, located on chromosome X, position 11.3, breaks down neurotransmitters which help control emotion (Buckholtz, et al., 2008). If identified, a better understanding of the genetic causes of aggression will result (Siever, et al., 2008). The MAOA gene was amplified using polymerase chain reaction (PCR) and identified by gel electrophoresis in humans (Homo sapiens) and zebra finches (Taeniopygia guttala). For the human experiment a forward primer (5'-AAT TGC CTG GTC TCC CCC AA-3') and a reverse (5'-ATT GTG TCT GCC TCC CCC AG-3') were used (Shumay et al., 2012). Annealing temperatures were set at 95℃ for denaturation, 60℃ for annealing, and 72℃ for the expansion phase (Shumay et al., 2012). We hypothesize that, the primers will anneal at 43,398,598 bp and 43,400,807 bp on the X chromosome, and amplify a DNA strand of 2,270 bp long showing the presence of the MAOA gene (Shumay et al., 2012).We hypothesize that the primers, forward (5' TAC TCG CCT TCT CCT GAC TC 3') and reverse (5' CCC AAA GCA TTC AAG ACC TCA C 3'), will anneal at 855 bp and 2270 bp onto the MAOA gene producing a band of 1415 bp long representing the presence of the MAOA gene (Livak, 2002). After experimentation, the MAOA gene was not successfully identified in humans as well as the zebra finch. However we discovered that the MAOA gene is a conserved gene across different species and should be researched further in order to better understand its role in aggressive behaviors across multiple organisms.
Written by: B315
Revised by: B200
Finalized by: B125
Experiment Summary
The MAOA gene, also known as the "warrior gene" because of its association with aggression is one of the most investigated genes in psychiatry (Shumay et al., 2012). It is located on chromosome X position 11.3, and codes for the monoamine oxidase (MAO) enzyme (Powledge, 2016). The monoamine oxidase enzyme is responsible for breaking down the neurotransmitters, dopamine, serotonin and norepinephrine (Powledge, 2016). Due to the MAOA-VNTR polymorphism in the gene, there are two alleles of the MAOA gene: higher MAOA (MAOA-H) or lower MAOA (MAOA-L) (Dorfman et al., 2016). The MAOA-L produces less of the MAO enzyme while the MAOA-H produces a normal amount (Dorfman et al., 2016). Having a mutation of the MAOA-L allele is what causes aggressive behavior in humans due to less uptake of serotonin, dopamine, and norepinephrine to the brain (Dorfman et al., 2016). The research conducted within this paper was to positively identify the MAOA gene in humans and a zebra finch homolog by using PCR and gel electrophoresis.
Homo Sapiens MAOA gene PCR and Gel Electrophoresis
With the use of PCR, DNA from a human epithelial cell was amplified and then gel electrophoresis was used to determine if the wild type MAOA gene was present in the test subjects. We predicted that with the forward primer, 5'-AAT TGC CTG GTC TCC CCC AA-3' and the reverse primer, 5'-ATT GTG TCT GCC TCC CCC AG-3', the MAOA gene would be successfully identified at 2,270bp as previously performed by Elena Shumay (Shumay et al., 2012). The procedure used by Elena Shumay was mimicked; therefore, we expected to attain similar results. We hypothesized that the primers will be at 43,398,598bp and 43,400,807bp on the X chromosome, and amplify a DNA strand that is 2,270 base pairs long to show the presence of the MAOA gene in humans.
Taeniopygia Guttata MAOA gene PCR and Gel Electrophoresis
The MAOA gene is found among aggressive and nonaggressive species (Figure 8). It can be stated that the MAOA genes’ amino acid sequence is significantly similar between these two types of organisms (aggressive and nonaggressive). This is because the amino acid sequence of domestic cats (Felis catus) and leopards (Panthera pardus) are almost identical. The process of amino acid sequencing was repeated for humans (Homo sapiens), chimpanzees (Pan troglodytes), and orangutans (Pongo abelii) to identify if they were transcribed in the Hominidae family. These animals were chosen in order to identify if the MAOA gene was present across species of the same family, and the results show that the gene is highly conserved. These animals were chosen based on their behavioral tendencies. For example, in cats and leopards were chosen because previous research showed that domesticated animals are aggressive significantly less frequently than non-domesticated organisms (Bradshaw and Cameron-Beaumont, 1988). Chimpanzees and orangutans were chosen because chimpanzees have been known to be more aggressive (Zucker, 1987). Compared to orangutans which are less aggressive due to their mating habits and social structure. (Wilson, 2014) This provides support that the MAOA gene is a conserved.
Nucleotide sequences were gathered from NCBI BLAST for the MAOA gene of different orders of species to discover if it was an interspecies gene. The animals were chosen and then their nucleotide sequences were compared to find similar nucleotides (Figure 5). The MAOA gene was discovered in multiple species which consisted of humans (Homo sapiens), Western Gorillas (Gorilla gorilla), Nine Banded Armadillo (Dasypus novemcinctus), Southern White Rhinoceros (Ceratotherium simum simum), and Sperm Whales (Physeter macrocephalus) (Dereeper, 2008).
We hypothesize that the primers will anneal 855 bp into the MAOA gene, and 2270 bp into the MAOA gene to produce a band that is 1415 bp long to show the presence of the MAOA gene in zebra finch. After running the nucleotide sequences, it can be stated that the MAOA gene is significantly similar among the five species therefore the forward (5' TAC TCG CCT TCT CCT GAC TC 3') and reverse (5' CCC AAA GCA TTC AAG ACC TCA C 3') primers generated, shown by the green highlighted sequences with the labels forward and reverse, based on their direction (Figure 5) (Kocker, 1989).
Controls
During experimentation a positive and negative control was used. A successful positive control is needed to indicate the PCR procedure was successful. Having a successful PCR procedures allowed us to identity the MAOA gene in both human and homolog. E.coli was used as the positive control and ddH2O was used as the negative control (Figure 1)(Figure 2). A PCR cocktail was created and the only variable that changed was the DNA added. In doing so, only annealing temperatures and primers were the variables that would cause success or failure during experimentation.
Figure 1 shows E. coli was amplified using polymerase chain reaction. A semi-log plot was constructed and the equation y=90655x^-3.306 was used to determine 168bp of E. coli. This indicates an unsuccessful trial as the accepted base pair length for E. coli is 520bp. The annealing temperature was changed from 58℃ to 60℃ for the primers to anneal more specifically to the DNA.
Figure 2 shows a gel where E.coli DNA was only loaded into wells two and six but it was seen in multiple wells thus indicating the gel was two thick and caused cracking and leakage. To avoid the gel from cracking a thinner gel was used. The streaking of the bands indicates that the 100V used should be decreased to 85V.
Unsuccessful Identification of the MAOA gene in Homo Sapiens and Taeniopygia Guttata
Amplification of the MAOA gene was not successful from isolated Homo Sapiens using the published primers from Shumay et al., 2012 (Figure 4). Using the semi-log plot, the positive and negative controls were denoted successful. Therefore, since published primers were used it can be concluded that the MAOA gene was not successfully amplified due to the set annealing temperatures. Although well three shows streaking, E.coli was 566bp which indicates success because the accepted value is 520bp (Paton et al., 1998). Well two indicates nonspecifically annealing as bright streaks begin above 100bp showing that the MAOA gene was not successfully amplified in human. This streaking was due to large amounts of nonspecific annealing. There was no contamination problems as indicated in well four. A gradient will be run in order to discover the best annealing temperature for the human DNA.
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Written by: B125
Revised by: B180
Finalized by: B200
Figure 2: Gel Electrophoresis of E. coli Positive Control. E. coli was amplified using polymerase chain reaction. The 49 µl of master mix was created with 40 µl of distilled water, 5 µl of 1x TBE of PCR buffer, 1 µl dNTPs, 1 µl of 8F forward primer, 1 µl of 529R reverse primer, and 1 µl of Taq polymerase. The PCR tubes were placed in a holding rack and 49 µl of master mix and 1 µl of test subject DNA were added into individual tubes which were then placed into the thermocycler. The PCR was run at 95℃ for 5 minutes, then cycled 30x at 95℃ for 1 minute, 61℃ for 1 minute and 30 seconds, and 72℃ for 1 minute, and finally 72℃ for 5 minutes. The PCR products then underwent gel electrophoresis. To create the agarose gel in erlenmeyer flask, 5 mL of 10x TBE and 45 mL of water were added to make 1x TBE. This was mixed with 0.5g of agarose powder which was then microwaved in intervals of 30 seconds for 1-3 minutes until the solution was clear. After letting it cool, 1.7 µl of 10,000x SYBR Green dye (indicator) was added to the flask and the gel tray and comb were prepared to hold the gel. The agarose gel was then coated with a buffer solution of 1x TBE until a thin layer of solution covered the top of the gel. 14 µl of ddH2O, 4 µl of 1kb plus ladder, and 2 µl of 10x blue loading dye were added into well one to make the ladder. Well two contained 18 µl of E. coli PCR product and 2 µl of 10x blue loading dye, while well three contained 18 µl of the ddH2O PCR product and 2 µl of 10x blue loading dye. Well four contained the 9 µl of E. coli PCR product and 1 µl of 10x blue loading dye. The voltage of the energy supply was set to 100V for 30 minutes. Using ultraviolet light, the gel was analyzed. A) Well one contained the 1kb Plus ladder. Wells two, three, and four, all showed faint streaking with a bright bands at the same point, just above the 500 base pair mark. There were also cracks in the gel, as the E. coli results were found in the water control, and the well next to well four. B) Migration distance (cm) vs. molecular size of 1kb Plus ladder was imputed to form a semi-log plot. Migration distances (cm) were measured using a ruler. This plot was then used to determine the base pair lengths of the E. coli bands shown. The equation of the line was placed into the graph (y=25933e-1.057x). The E.coli travelled 3.6 cm which was inputted into the equation to show the band was 577 base pairs long. Since the E. coli length using these primers supposed to be about 500 base pairs, it was concluded that the E. coli positive control was successfully amplified under these conditions. Troubleshooting included decreasing the voltage of the energy supply to about 85V to decrease streaking, and to make a thinner gel.