Discovery of Tryptophan Hydroxylase 1 Gene in Homo Sapiens and Class Actinopterygii Using PCR & Gel Electrophoresis

Lauren Eby, Allison Loch, Jenny Ward, and Riley Woerner

Abstract

The tryptophan hydroxylase 1 gene (TPH1) codes for the tryptophan hydroxylase enzyme, which regulates serotonin levels in the brain. In the A218C mutation, the gene sequence contains a base pair change, which leads to an increased incidence of suicidal behaviors among the population. Other mutations in the TPH1 sequence have been attributed to suicidal ideation and behaviors (Beden et al, 2016). PCR was used with site-specific primers to target and amplify the wild-type TPH1 sequence. The successful amplification of the TPH1 gene in the human, zebrafish, sturgeon, and trout genomic DNA sequence was verified through gel electrophoresis. We hypothesized that by using published primers, we will successfully amplify a 5770 base pair product from the Homo sapien sequence using PCR and gel electrophoresis
because the forward primers will anneal at base pair 65 and the reverse primer will anneal at base pair 5,835. Additionally, we hypothesized that by designing site-specific PCR primers based on conserved sequences in the TPH1 gene of the zebrafish sequence, a 706 base pair product will be yielded for Danio rerio, Acipenser fulvescens, and Salvelinus manaycush because the primers
were designed from conserved sequences within the zebrafish genomic sequence, which has high homology with the other organisms, and will anneal at base pair 5 and base pair 709. The Qiagen Blood and Cell Culture Mini Kit was first utilized to unsuccessfully purify human DNA collected in Oragene DNA collection kits for saliva. The Qiagen Generation Capture Column Kit was successfully used to obtain a yield of up to 3,990 nanograms of genomic DNA at 1.86 purity ratio. Lambda phage was used as a positive control and dye was used as a negative control in all experiments. PCR was used to test the presence of the TPH1 gene in the human, zebrafish, sturgeon, and trout genomic DNA, which was then verified with gel electrophoresis. A sequence was not amplified in the human or trout PCR trials. Bands of 690 and 703 base pairs, were visualized from the zebrafish and sturgeon PCR cocktails, suggesting that the TPH1 gene was successfully amplified in these samples.

Discussion

The tryptophan hydroxylase 1 gene is integral in the regulation of the release of serotonin: the wild type TPH1 gene codes for the TPH enzyme which catalyzes the release of serotonin (Beden et al, 2016). Serotonin is important for maintaining an overall sense of well-being, and if not regulated properly, can become associated with suicidal behavior, impulsive aggression, antisocial behavior, obsessive-compulsive disorder, anxiety disorders, and alcoholism (Mann, 1999). Mutations in the TPH1 gene can lead to problems in serotonin regulation, which could lead to suicidal behaviors (Liu and Quinn, 2006). Suicide is the third leading cause of death in the world, accounting for nearly one million deaths annually; suicide is also the leading cause of death for people ages 15-44 (Gonzalez-Castro et al, 2014). Research has been conducted on the wild type TPH1 gene to acquire a higher level understanding of how the TPH1 gene affects and regulates serotonin (Gonzalez-Castro et al, 2014).

PCR can be used to amplify specific sequences of a gene that are of interest to the researchers, and gel electrophoresis is used to visualize the amplified DNA in the form of a band that corresponds to the base pair length of the target DNA (Liu and Quinn, 2006). Therefore, we hypothesized that because the published primers that we used were designed to anneal and amplify the A218 allele in the wild type TPH1 gene, we expected to see a band that corresponds to the base pair length of the A218 allele.

After studying a human’s TPH1 wild type sequence the question arose: is there an animal that exists that has a homologous genetic sequence that could potentially be linked to a similar behavior as the one we see in humans? Based on this question we hypothesized that after using PCR to detect a homologous genetic sequence in a zebrafish, the band in gel electrophoresis would be comparable to that of human’s, because of the similarities of genomic sequences in the TPH1 genes (Rujescu et al, 2002).

In order to become acquainted with optimal conditions and temperatures suitable for PCR, as well as develop a control for later experiments, an initial PCR reaction was conducted utilizing the lambda virus Rz1 gene. Numerous experimental trials were carried out, during which Taq polymerase quantities, buffer amounts, and buffer types were adjusted to obtain the best results (Abilock and Stephenson, 2012). This experiment served as a control, as we successfully obtained a band at 400 base pairs in the gel. We knew that the lambda virus could accurately be run through PCR, and if a problem arose in a different cocktail, it was due to that cocktail and not the gel.

Through the use of PCR and gel electrophoresis techniques, we were not able to amplify the A218 target allele in human DNA. We believe that this is due to the use of fragmented human DNA. However, we utilized the same PCR and gel electrophoresis methods to seek out a homologous genetic sequence in zebrafish, and later, sturgeon. We designed primers that annealed to a sequence in the zebrafish DNA that is similar to the human sequence (Rujescu et al, 2002). By finding this homologous genetic sequence, we may be able to find a behavior in the zebrafish that is similar to the novel human behavior that is regulated by the TPH1 gene (Rujescu et al, 2002). Further research can be done on the homolog and later be applied back to the human gene and behavior (Gonzalez-Castro et al, 2014). This comparison is important, as previous and extensive research has been done that has been focused on the TPH1 gene in humans, and the behaviors that are controlled by this gene (Gonzalez-Castro et al, 2014). However, little to no research has been done on the TPH1 gene in other organisms, such as zebrafish and sturgeon. This research can further validate the relationship between the TPH1 gene and the novel behaviors that are controlled by it (Mann, 1999).

When performing our PCR and gel electrophoresis control experiment with the lambda virus, there were certain weaknesses in our experimental design that initially set us back. At first, we were not using the correct proportions of PCR buffer and Taq polymerase in our reaction cocktail. We were utilizing too much Taq polymerase, and not enough buffer. Additionally, we were using a PCR buffer that did not contain magnesium chloride. The magnesium chloride is important for the annealing step of the PCR reaction: the magnesium chloride acts as a cofactor to the Taq polymerase and is crucial for the binding specificity of the primers (Abilock and Stephenson, 2012). These flaws were then corrected by adjusting the amounts of the buffer and Taq polymerase and making sure that a buffer that contained magnesium chloride (MgCl₂) was added to the PCR cocktail. With the magnesium chloride present, and amounts adjusted accordingly, the PCR reaction worked correctly, as signified by the appearance of a band at 420 base pairs during gel electrophoresis in the Rz1 Lambda virus gene (Abilock and Stephenson, 2012).

In addition to flaws in the PCR portion of our experiment, there were several minor flaws in our gel electrophoresis. During our first trial run of the PCR cocktail through the gel, we set our voltage much too low. As a result, the DNA did not run and was stuck in the well. This was corrected by setting the voltage to an intermediate level (135V) and running it for a short amount of time (Abilock and Stephenson, 2012). After it ran for a brief time, we would check to make sure that the DNA and 1Kb Plus DNA Ladder were running through the gel correctly. Then we would continue at the same voltage and run the gel for about 15-20 minutes to completion (Abilock and Stephenson, 2012).

Since the TPH1 gene was successfully amplified in zebrafish and sturgeon, we would like to amplify it in other organisms of the Actinopterygii class shown in Figure 5. Determining its existence in these organisms could help us determine the evolutionary pathway of the TPH1 gene and add more detail to our phylogenetic tree. In addition to this, the genomic sequences of the gene in the different organisms could be mapped and compared.

Much work was done on the genotypic attributes of these organisms. We would like to do more exploration on the phenotypes of this gene. It is known that in humans, mutations in the TPH1 gene can lead to problems in serotonin regulation, which could lead to suicidal behaviors (Liu and Quinn, 2006). As such, studying the behavioral aspects of the gene in other organisms could lead to a better understanding of how it impacts us, which would be extremely valuable since it could potentially save lives.

We would like to successfully amplify the TPH1 gene using human DNA. If given the proper time and resources to do this, we would use the QIAGEN Blood and Cell Culture Mini Kit with blood cells. We believe that it would be easier to acquire the necessary quantities and purities of DNA from blood, since only about 5% of spit contains cheek cells. We would also consider redesigning our primers to amplify a smaller section of the TPH1 gene. Amplifying a smaller sequence could make the PCR process more efficient.

Figure 8: Gel electrophoresis visualization of PCR amplified Sturgeon TPH1 gene. (a) A target sequence of roughly 700 base pairs was amplified using the forward designed primer of 5’-GGTTAAAAGACCCAGGGCGT-3’ and the reverse designed primer of 5’-AAAGCCTTGACTAGCCCACC-3’. The PCR cocktail was composed of 36 μL nuclease-free water, 7 μL 10X PCR buffer, 2 μL MgCl2, 1 μL of Taq polymerase, 1 μL dNTP’s, 1 μL lake sturgeon DNA, and 1 μL of both forward and reverse primers. An additional cocktail was made using the same ingredients, and substituting the Lake Sturgeon DNA for 1 μL zebrafish DNA template. A thermocycler was used to run the cocktail for 30 cycles. The PCR cycles ran for 10 min at 95℃ for one initial denaturation step, and then 30 cycles of alternating 30s at 95℃ for denaturation, 1 min at 57℃ for annealing, and 1 min at 72℃ for elongation. The final elongation step was 72℃ for 7 mins (Kim, 2009). A 0.8% agarose gel made with TBE, agarose powder, and GloGreen dye was used for gel electrophoresis. 5 μL of 1 Kb Plus DNA Ladder was pipetted into well 1. 10 μL of the various PCR cocktails containing designated DNA template were mixed with 5 μL of loading dye for pipetting into wells. Well 2 contains the lambda Rz1 gene cocktail. Well 3 contains the zebrafish cocktail with MgCl2. Well 4 contains the sturgeon cocktail without MgCl2. Well 5 contains the zebrafish template with MgCl2. Well 6 contains the sturgeon cocktail with MgCl2. Well 7 contains a negative control of dye. The apparatus was run at 135 V for approximately 20 minutes. (b) Semi-log Graph used to analyze PCR products of the Zebrafish and Sturgeon TPH1 gene amplification. Migration distance (cm) of the bands of the ladder away from the well is represented by the x-values, and the molecular size of the DNA molecules (base pairs) is represented by the y-values. A logarithmic trendline produced an equation visualized in the graph. The equation was used to determine the base pair length of the lake surgeon DNA PCR product; it was calculated that the band appeared at 703.4 base pairs by inputting the 4.2 cm travelled from the middle of the well to the top of the band produced for the x in the equation. The y-value produced is the base pair length of the amplified DNA. The positive control of Lambda migrated 4.7 cm and was 419.9 base pairs in length.

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