Finalized by: B185

      Monoamine Oxidase A (MAOA) is a gene commonly associated with aggressive behaviors in many homologs when the low activity allele of the gene is present (MAOA-L). This study attempted to identify and clone the MAOA gene in humans, but first PCR and gel electrophoresis was used to correctly identify a segment of enterobacteria phage lambda at exactly 395 base pairs. The human MAOA replication attempt was done using DNA from human cheek cells, polymerase chain reaction, and agarose gel electrophoresis. The primers and annealing temperatures used for PCR were found from previous research (Shumay et al., 2012). We hypothesized that performing PCR with FPrimer1 and RPrimer1 and an annealing temperature of 60℃ would correctly amplify the human MAOA genome (Shumay et al., 2012). Although the primers did not result in an amplified human MAOA segment, we hypothesized that by comparing the genomic sequences of several organisms, primers could be created for future PCR tests in which would allow for the identification of a conserved MAOA gene in the Galapagos Sea Lion using gel electrophoresis (Siepel et al., 2005). If the gene is present, the created primers should result in a successful amplification of the gene. The significance of these findings would provide a better understanding of how many different organisms MAOA is present in allowing conclusions to be made in which prove its importance and evolutionary background (Siepel et al., 2005).

Finalized by: B110

      The MAOA gene, commonly referred to as the warrior gene, is responsible for an enzyme that degrades amine neurotransmitters including dopamine, noradrenalin and serotonin (Hunter, 2010). The warrior gene is linked to many psychiatric disorders including antisocial behavior, increased aggression, depression, and even autism (Cohen et al., 2011, Sabol et al., 1998, Frazzetto et al., 2007, Du et al., 2004). MAOA-L, the low activity allele, is linked to low IQ and aggression whereas in MAOA-H, the high activity allele, is shown to be be associated with depression (Meyer et al., 2006). The question addressed in this study was if primers could be designed to amplify the MAOA gene in the Galapagos sea lion using PCR. We hypothesized that if the MAOA gene exists in the Galapagos sea lion, and PCR is performed with primers designed from aligning the genomes of four different organisms containing the MAOA gene, then the primers will anneal correctly and amplify the targeted segment of the genome (Figure 2)(Karere et al., 2012).

Human MAOA gene PCR and Gel Electrophoresis

      We predicted the MAOA gene for humans will be successfully amplified using the FPrimer1 and RPrimer1 and an annealing temperature of 60℃ because this has been successfully been done in published research, with a band occurring at 444 bp when run through agarose gel electrophoresis (Shumay et al., 2012). By replicating the methods used by Shumay it is expected to have similar results. We hypothesize that conducting PCR with FPrimer1 and RPrimer1 and using an annealing temperature of 60℃ will result in correct amplification of the MAOA gene at 444 base pairs when run through gel electrophoresis (Shumay et al., 2012). Understanding this data and being able to isolate the MAOA gene can be used to help diagnose those with MAOA-L or MAOA-H alleles (Widom and Brzustowics, 2006).

      The amplification of published human primers on human DNA was deemed unsuccessful (Figure 8). Since the control, lambda, was successfully amplified it can be deduced that the mastermix was not the cause of unsuccessful amplification of the human MAOA gene. Since the primers were published the likely problem as to why the human MAOA gene was not amplified was because the researchers used a gradient, 50-58℃, on their annealing temperatures for their PCR aliquots. The assays annealing temperature was set at 55℃ which could have been too high for the primers to correctly anneal onto the DNA template.

Galapagos Sea Lion MAOA gene PCR and Gel Electrophoresis

      We predict that the MAOA gene will be able to be identified in the Galapagos sea lion through the use of PCR and gel electrophoresis because the MAOA gene is conserved among many organisms. An organism that has many physiological and genetic similarities to the Galapagos sea lion, the Weddell seal, has been found to have the MAOA gene (Boness, 1991)(Arnason et al., 2006). The Weddell seal and Galapagos sea lion are in the same suborder of Carnivora, called Caniformia. This relationship is a closer than the relation between humans and mice, which are both found to have the MAOA gene with 100% conservation (Figure 1)(Dereeper et al., 2008). By knowing that the MAOA gene exists in a similar organism, it is plausible that it also exists in the Galapagos sea lion. We hypothesize that if the MAOA gene exists in the Galapagos sea lion, that the designed primers, from conserved parts of the genome of four different animals, will anneal to their complementary DNA strands and the MAOA gene will be able to be amplified (Karere et al., 2012).

      The designed homolog primers were used on extracted human DNA, because Galapagos sea lion DNA was not acquired for testing. The PCR cocktail with homolog primers and human DNA, E. coli control, and ladder was ran through gel electrophoresis. The resulting gel showed no band for the MAOA gene and the control did not leave the well (Figure 7). This was inconclusive of whether or not the primers were correctly designed and was an indicator that we needed to switch our control back to the lambda virus.

Controls

      The amplification of the bacteriophage lambda virus and E. coli using PCR acted as a positive control for all other PCR experiments performed throughout the study (Figure 2)(Figure 3). In the beginning of the study the Lambda virus was the control, but due to lack of resources the control was switch to E. coli. Since E. coli resulted in unsuccessful amplification after multiple trials the control was switched back to lambda. Each PCR cocktail was a mastermix meaning the same taq polymerase, nucleotides and PCR buffer were used in all PCR cocktails created that day. This reduced the amount of variability in the PCR cocktails by ensuring that the variation in the results of running gel electrophoresis is due only to the differences in primers and the DNA used.

Potential Error, Experimental Constraints, and Possible Solutions

      Error can display itself in many different ways while performing PCR and agarose gel electrophoresis. In order to more accurately determine the base pair lengths of the isolated DNA sequence, a semi-log plot was crafted. The equation generated from the semi-log plot was used to estimate the length of the isolated sequence. This prevented inaccurate estimations based on the visual appearance of the unknown band's location in relation to the known DNA lengths of the molecular weight ladder. All calculations of experimental base pair lengths, from the semi-log plot, are given a 10% margin of error to be accepted. This is because the process of gel electrophoresis is not always entirely reliable and variation in DNA sequences can contribute to variance in base pair length (Van Donkersgoed et al., 2001). Photos of gels were taken immediately using the Bio Rad Gel Doc EZ Imager after it was run through gel electrophoresis. This ensured our data was as accurate as possible because as gels sit the DNA diffuses into the gel, causing bands to lose their clarity. A semi-log plot was crafted using pixels as the running distance, rather than centimeters that were suggested because it added another level of accuracy by increasing the number of significant digits used.

      To determine if the MAOA was present in the Galapagos sea lion, it was crucial to run the homolog primers on Galapagos sea lion DNA. By not obtaining the Galapagos sea lion DNA a large constraint was placed on the study. The designed primers were tested on human DNA template but the trial was unsuccessful due to an insufficient amount DNA present in the PCR cocktail. This resulted in an incomplete conclusion on whether the designed primers were capable of targeting the MAOA gene.

      Even though the primers for the Galapagos sea lion were never officially proven wrong, potential error could have occurred in our primer design. When creating the primers for the homolog organism, organism genomes with known MAOA sequences were aligned to find similarities. The error could be from choosing four random species rather than picking organisms closer in evolutionary distance (Berezikov et. al, 2005). This would eliminate more factors of genomic differentiation that can occur over time as organisms evolved farther away from each other.

      Originally the lambda virus was used as the control, but due to lack of resources the control was switched to E. coli. The assays with E. coli was not successful because the DNA would remain in the well. Since the whole genome remained intact, the primers 8F and 529R did not anneal to the DNA template. The PCR cocktail consisted of the same ingredients and concentrations as used in previous successful trials and the annealing temperature was set to a gradient from 50-58℃. This eliminated the suspicion of the PCR cocktail and PCR cycles from being the issue. If there was more time to complete this study, more Lambda DNA and primers would have been purchased to continue the use of it as a control.

      The reason why the MAOA gene in the assay with human DNA template and the homolog primers had no band was because human DNA had insufficient amount of template in the PCR cocktail. To save time the DNA sample was immediately ran through PCR before the analytical gel was finished for genome prep. When the genomic prep analytical gel was finished there was no DNA in the gel, which is why the PCR was unsuccessful. In the future to prevent this from happening, complete analyzations of tests should be completed before moving forward.

      Genomic prep was repeated many times because extracted DNA was not prevalent when ran through gels. The QIAGEN genomic prep kits required many buffers which were mixed up often, causing the extracted DNA to become lost in waste. After many misfires with the QIAGEN kit, Chelex 100 beads were purchased. With the new kit and method of chewing cheeks before spit collection, a higher mass of DNA was extracted, 0.203 mg. In the future Chelex 100 beads should be the first resort for genomic prep because they were more effective in DNA extraction and was time efficient.